Alkyl chain modified imidazoquinoline TLR7/8 agonist compounds and uses thereof

ABSTRACT

Disclosed are alkyl chain modified 1H-imidazoquinoline compounds, derivatives and analogs thereof, as Toll-like receptor-7 and -8 agonists for enhancing immune responses. Also provided are methods of making pharmaceutical compositions containing these compounds. The present disclosure also describes methods of use for the alkyl chain modified 1H-imidazoquinoline compounds, derivatives and analogs thereof, and pharmaceutical compositions containing these compounds for the treatment of disease in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 16/107,605, filed Aug. 21, 2018, which claims priority to andthe benefit of U.S. Provisional Patent Application No. 62/548,848, filedAug. 22, 2017, the disclosures of each of which are incorporated hereinby reference in their entireties.

FIELD OF THE INVENTION

The present disclosure relates to alkyl chain modified imidazoquinolineTLR7/8 agonist compounds for enhancing immune responses. The presentdisclosure also relates to pharmaceutical compositions comprising thealkyl chain modified imidazoquinoline compounds, methods of preparationthereof, methods for stimulating an immune response, and uses of thepharmaceutical compositions in the treatment of disease in a subject,e.g., infectious disease and cancer.

BACKGROUND OF THE INVENTION

Toll-like receptors (TLRs) are a family of transmembrane proteins thatrecognize structurally conserved molecules that are derived from andunique to pathogens, referred to as pathogen-associated molecularpatterns. As such, TLRs function in the mammalian immune system asfront-line sensors of pathogen-associated molecular patterns, detectingthe presence of invading pathogens (Takeuchi and Akira 2010 Cell140:805-820). TLR engagement in sentinel immune cells causesbiosynthesis of selected cytokines (e.g., type I interferons), inductionof co-stimulatory molecules, and increased antigen presentationcapacity. These are important molecular mechanisms that activate innateand adaptive immune responses. Accordingly, agonists and antagonists ofTLRs find use in modulating immune responses. TLR agonists are typicallyemployed to stimulate immune responses, whereas TLR antagonists aretypically employed to inhibit immune responses (Gosu et al 2012Molecules 17:13503-13529).

The human genome contains 10 known TLRs, of these TLR3, TLR7, TLR8, andTLR9 sense nucleic acids and their degradation products. Thedistribution of TLR7, TLR8, and TLR9 is restricted to the endolysosomalcompartments of cells and they are preferentially expressed in cells ofthe immune system. In the activated, dimeric receptor configuration TLR7and TLR8 recognize single stranded RNA at one ligand binding site andthe ribonucleoside degradation products guanosine and uridine,respectively, (as well as small molecule ligands with related structuralmotifs) at a second ligand binding site (Zhang et al 2016 Immunity45:737-748; Tanji et al 2015 Nat Struct Mol Biol 22:109-115). Engagementof TLR7 in plasmacytoid dendritic cells leads to the induction ofinterferon-α/β, which plays essential functions in the control of theadaptive immune response (Bao and Liu 2013 Protein Cell 4:40-52).Engagement of TLR8 in myeloid dendritic cells, monocytes, andmonocyte-derived dendritic cells induces a prominent pro-inflammatorycytokine profile, characterized by increased production of tumornecrosis factor-α, interleukin-12, and IL-18 (Eigenbrod et al 2015 JImmunol 195:1092-1099). Thus, virtually all major types of monocytic anddendritic cells can be activated by agonists of TLR7 and TLR8 to becomehighly effective antigen-presenting cells, thereby promoting aneffective innate and adaptive immune response. Most antigen presentingcell types express only one of these two receptors, accordingly smallmolecules with potent agonist bioactivity against both TLR7 and TLR8receptors are potentially more effective immune adjuvants than agonistsspecific for only one of these TLRs. Thus a TLR7/TLR8 (TLR7/8) smallmolecule agonist with balanced, dual bioactivity would cause innateimmune responses in a wider range of antigen presenting cells and otherkey immune cell types, including plasmacytoid and myeloid dendriticcells, monocytes, and B cells (van Haren et al 2016 J Immunol197:4413-4424; Ganapathi et al 2015 PLoS One 10:e0134640). Such potentdual TLR7/8 agonists may also be effective in stimulating effectiveanti-tumor immune responses in cancer (Singh et al 2014 J Immunol193:4722-4731; Sabado et al 2015 Cancer Immunol Res 3:278-287; Spinettiet al 2016 Oncoimmunol 5:e1230578; Patil et al 2016 Mini Rev Med Chem16:309-322).

A number of small molecule structural classes are known to interact atthe guanosine/uridine ligand binding site and possess varying levels ofTLR 7 and/or TLR8 agonist bioactivity (see e.g., Lu et al 2012 ClinCancer Res 18:499-509; U.S. Pat. Nos. 5,446,153, 6,194,425, 6,110,929,and 7,199,131), including derivatives of 1H-imidazo[4,5-c]quinoline thatare TLR7 agonists or dual TLR7/8 agonists (see e.g., Vasilakos and Tomai2013 Expert Rev Vaccines 12:809-819; U.S. Pat. No. 4,689,338). One such1H-imidazo[4,5-c]quinoline is1-isobutyl-1H-imidazo[4,5-c]quinolin-4-amine (Imiquimod), aTLR7-specific agonist that was approved in 1997 for the treatment ofactinic keratosis, superficial basal cell carcinoma, and genital warts,and was subsequently approved for the treatment of basal cell carcinoma(see e.g., Hemmi et al 2002 Nat Immunol 3:196-200). While some1H-imidazo[4,5-c]quinolines display selective TLR7 or TLR8 agonistactivities, others display dual TLR7/8 agonist activities. For example,1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine was found to be aTLR7 agonist with negligible bioactivity against TLR8 (Shukla et al 2010J Med Chem 53:4450-4465). In contrast,2-propyl[1,3]thiazolo[4,5-c]quinolin-4-amine was found to be a TLR8agonist with negligible activity against TLR7 (Gorden et al 2005 JImmunol 174:1259-1268).1-(4-aminomethylbenzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (IMDQ)and 1-(3-aminomethylbenzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine(meta-IMDQ) were found to be dual TLR7/8 agonists with potent agonistactivity against both receptors (see e.g., Shukla et al 2010 J Med Chem53:4450-4465; Shukla et al 2010 Bioorg Med Chem Lett 10:6384-6386; U.S.Pat. Nos. 8,728,486; 9,441,005).

However, rapid systemic distribution of soluble1H-imidazo[4,5-c]quinoline-based TLR7/8 agonists following subcutaneous,intratumoral or intramuscular administration has been demonstrated tocause significant toxicities in patients (see e.g., Vasilakos et al 2013Expert Rev Vaccines 12:809-819; Savage et al 1996 Br J Cancer74:1482-1486; Pockros et al 2007 J Hepatol 47:174-182). Systemic immunesystem activation due to activation of TLRs in cells of the spleen andliver causes an increase in serum pro-inflammatory cytokine levels,which in turn causes flu-like symptoms and other adverse events thatlimits the utility of these compounds as human therapeutics to a topicalroute of administration. Thus, there remains a need for small moleculetherapeutic agents with potent and balanced TLR7/8 agonist activitiesthat also possess physiochemical properties that enable pharmaceuticalcompositions that promote retention of the compound at the site ofinjection.

BRIEF SUMMARY OF THE INVENTION

The present disclosure provides alkyl chain modified1H-imidazo[4,5-c]quinoline derivatives that are potent TLR7/8 agonistsexhibiting balanced bioactivity against both receptors. In one aspect,provided is a compound of formula (J):

or a salt thereof, wherein:

R⁰ is C₄-C₂₁ hydrocarbyl optionally substituted by 1 to 4 halogen atoms;

X is —NH— or —NH(C═O)—;

R¹ is C₃-C₆ alkyl, —(CH₂)_(p)OR^(1a), —(CH₂)_(p)NHR^(1b), or—(CH₂)_(p)R^(1c); where R^(1a) and R^(1b) are independently C₁-C₃ alkyl;R^(1c) is C₃-C₄ cycloalkyl; and p is 1 or 2;

R² is NHR^(2a); where R^(2a) is H, OH, NH₂, or methyl;

each R³ is independently halogen, C₁-C₈ alkyl, —(C₁-C₇ alkylene)—NH₂, or—CH₂-phenylene-CH₂NH₂;

q is 0, 1, 2, 3, or 4; and

R^(4a) and R^(4b) are independently H or C₁-C₈ alkyl,

provided that the compound is other than2-butyl-1-(4-((hexadecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine(Compound No. 63-32);N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)palmitamide(Compound No. 63-31); orN-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)pent-4-ynamide(Compound No. 63-37).

In some embodiments, R⁰ is C₄-C₁₄ hydrocarbyl.

In some embodiments, X is —NH(C═O)—. In some embodiments, X is —NH—.

In some embodiments, R⁰ is branched C₄-C₁₄ alkyl,—(CH₂)_(z)(C(CH₃)₂)R^(A), or —(CH₂)_(m)R^(A); m is 0, 1, 2, or 3; z is 1or 2; and R^(A) is C₃-C₈ cycloalkyl optionally substituted by 1 to 4groups independently selected from the group consisting of C₁-C₄ alkyland C₁-C₄ alkylene.

In some embodiments, R⁰ is branched C₄-C₁₄ alkyl.

In some embodiments, R⁰ is —(CH₂)_(m)R^(A). In one variation, m is 1 or2, and R^(A) is cyclopropyl, cyclobutyl, or cyclopentyl.

In some embodiments, R⁰ is —(CH₂)_(z)(C(CH₃)₂)R^(A). In one variation, zis 1, and R^(A) is cyclopropyl, cyclobutyl, or cyclopentyl.

In some embodiments, R^(A) is C₃-C₈ cycloalkyl.

In some embodiments, R^(A) is C₃-C₆ cycloalkyl optionally substituted by1 to 3 groups independently selected from the group consisting of methyland methylene. In one variation, m is 1 or 2.

In some embodiments, R^(A) is cyclopropyl optionally substituted by 1 to3 groups independently selected from the group consisting of methyl andmethylene, and m is 1 or 2.

In some embodiments, m is 0 or 1, and R^(A) is cyclohexyl optionallysubstituted by 1 to 3 groups independently selected from the groupconsisting of methyl and methylene.

In some embodiments, R⁰ is selected from the group consisting of:

In some embodiments, R¹ is C₃-C₆ alkyl (e.g., n-butyl). In someembodiments, R¹ is —(CH₂)_(p)OR^(1a) (e.g., CH₂OCH₂CH₃). In someembodiments, R¹ is —(CH₂)_(p)NHR^(1b) (e.g., CH₂NHCH₂CH₃). In someembodiments, R¹ is —(CH₂)_(p)R^(1c). In one variation, R^(1c) iscyclopropyl.

In some embodiments, R² is NH₂.

In some embodiments, q is 0. In some embodiments, q is 1 and R³ is C₁-C₈alkyl.

In some embodiments, each R^(4a) and R^(4b) is H.

In some embodiments, the compound is selected from the group consistingof Compound Nos. 63-33 to 63-36 and 63-38 to 63-49 in Table 1, or a saltthereof.

In another aspect, provided is a compound of formula (K):

or a salt thereof, wherein:

n is an integer from 4 to 21;

X is —NH— or —NH(C═O)—;

R¹ is C₃-C₆ alkyl, —(CH₂)_(p)OR^(1a), —(CH₂)_(p)NHR^(1b) or—(CH₂)_(p)R^(1c); where R^(1a) and R^(1b) are independently C₁-C₃ alkyl;R^(1c) is C₃-C₄ cycloalkyl; and p is 1 or 2;

R² is NHR^(2a); where R^(2a) is H, OH, NH₂, or methyl;

each R³ is independently halogen, C₁-C₈ alkyl, —(C₁-C₇ alkylene)—NH₂, or—CH₂-phenylene-CH₂NH₂;

q is 0, 1, 2, 3, or 4; and

R^(4a) and R^(4b) are independently H or C₁-C₈ alkyl,

provided that the compound is other than2-butyl-1-(4-((hexadecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine(Compound No. 63-32) orN-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)palmitamide(Compound No. 63-31).

In some embodiments, X is —NH—. In one variation, n is an integer from 4to 15. In another variation, n is 4, 5, 6, or 7.

In some embodiments, X is —NH(C═O)—. In one variation, n is 11, 12, 13,or 14.

In some embodiments, R¹ is C₃-C₆ alkyl (e.g., n-butyl). In someembodiments, R¹ is —(CH₂)_(p)OR^(1a) (e.g., CH₂OCH₂CH₃). In someembodiments, R¹ is —(CH₂)_(p)NHR^(1b) (e.g., CH₂NHCH₂CH₃). In someembodiments, R¹ is —(CH₂)_(p)R^(1c). In one variation, R^(1c) iscyclopropyl.

In some embodiments, R² is NH₂.

In some embodiments, q is 0. In some embodiments, q is 1 and R³ is C₁-C₈alkyl.

In some embodiments, each R^(4a) and R^(4b) is H.

In some embodiments, the compound is selected from the group consistingof Compound Nos. 63-01 to 63-30 in Table 1, or a salt thereof.

Further provided are pharmaceutical compositions comprising (i) acompound of formula (J) or (K) and (ii) one or more pharmaceuticallyacceptable excipients. In some embodiments, the pharmaceuticalcompositions further comprise an antigen. In some embodiments, thepharmaceutical composition is comprised of pharmaceutically acceptableexcipients that include USP-grade oils and an organic modifier (e.g.,95% sesame oil/5% ethanol). In some embodiments, the pharmaceuticalcomposition is comprised of pharmaceutically acceptable excipients thatenable an oil-in-water nanoemulsion or a liposomal formulation, examplesof which are known to those skilled in the art. In some embodiments, thepharmaceutical composition can include an admixture of an antigen orantigens, including but not limited to tumor associated antigens orneoantigens.

The present disclosure also provides a method of stimulating an immuneresponse in a mammalian subject in need thereof, comprisingadministering to the mammalian subject a pharmaceutical composition asdescribed above in an amount, at a frequency, and over a time framesufficient to stimulate an immune response in the mammalian subject. Inone aspect, the immune response is a local immune response. In anotheraspect, the immune response is a systemic immune response.

The present disclosure also provides a plurality of methods for using apharmaceutical composition described above in a mammalian subject, suchas a human patient. In one aspect, methods are provided for treatingcancer in a mammalian subject in need thereof, comprising administeringto the mammalian subject the pharmaceutical composition in an amountsufficient to treat cancer in the mammalian subject. In another aspectof the method, intratumoral delivery comprises injection of thepharmaceutical composition into at least one tumor lesion. In one aspectof the method, an effective amount of a second therapeutic agent isfurther administered to the subject. In certain embodiments, the secondtherapeutic agent is a chemotherapeutic agent, an epigenetic modulator,inducer of immunogenic cell death, or an antagonist of an inhibitoryimmune checkpoint molecule. In another aspect, methods are provided forinducing an antigen-specific antibody response in a mammalian subject inneed thereof, comprising administering to the mammalian subject thepharmaceutical composition in an amount sufficient to induce anantigen-specific antibody response and/or an antigen-specific T cellresponse in the mammalian subject. In one aspect, methods are providedfor treating or preventing an infectious disease in a mammalian subjectin need thereof, comprising administering to the mammalian subject thepharmaceutical composition in an amount sufficient to treat or preventan infectious disease in the mammalian subject. In one aspect, methodsare provided for treating or preventing an IgE-related disorder in amammalian subject, comprising administering the pharmaceuticalcomposition in an amount sufficient to treat or prevent an IgE-relateddisorder in the mammalian subject.

Also provided in the invention are kits comprising pharmaceuticalcompositions of the invention, and instructions for use in the treatmentof infectious diseases and/or cancers. Methods are also provided for themanufacture of kits for use in the treatment of infectious diseaseand/or cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B show the change in serum levels of IL-6 (FIG. 1A) andIL-12p40 (FIG. 1B) over time post injection following a singlesubcutaneous administration of Compound Nos. 63-00 (black bars), 63-17(white bars), and 63-10 (grey bars) to wild-type mice as described inExample B3. Group size=3, +/− standard error of the mean.

FIGS. 2A-C show tumor growth inhibition in syngeneic CT26 tumor bearingwild-type mice following repeated intratumoral administration ofCompound Nos. 63-18 (FIG. 2A), 63-33 (FIG. 2B), or 63-10 (FIG. 2C) asdescribed in Example B4. Animals were dosed as described with eithervehicle control (-●-), 20 μg of compound (-▪-), 5 μg of compound (- -▪--), 0.5 μg compound (--▪--), or 50 μg of a TLR9 agonist positive control(-♦-). Group size=5 for controls and 8 for experimental conditions, +/−standard error of the mean.

FIGS. 3A-B show tumor growth inhibition over time in the injected (FIG.3A) and distal (FIG. 3B) tumors of CT26 tumor-bearing wild-type micefollowing repeated intratumoral administration of pharmaceuticalcompositions comprised of a squalene-based oil-in-water nanoemulsionvehicle control (-●-), a squalene-based oil-in-water nanoemulsion with50 ng Compound No. 63-10 (-▪-), or a squalene-based oil-in-waternanoemulsion with 50 ng Compound No. 63-10 plus 50,000 ng AH-1 class IIpeptide (-□-) as described in Example B5. Animals were dosed asdescribed on experimental days 8, 12, 16, and 20. Group size=8 forcontrols and all experimental conditions, the data is expressed asaverage tumor volume (in mm³) +/− standard error of the mean.Differences in tumor volumes between groups on day 27 for the injectedtumor, or on day 23 for the distal tumor, were analyzed using aKruskall-Wallis test followed by Dunn's post-test for specific grouppair comparisons. ns indicates P≥0.050; * indicates P≤0.050.

FIGS. 4A-B show tumor growth inhibition over time in the injected (FIG.4A) and distal (FIG. 4B) tumors of CT26 tumor-bearing wild-type micefollowing a single intratumoral administration on experimental day 14 ofpharmaceutical compositions comprised of phosphate buffered salinevehicle control (-●-), phosphate buffered saline vehicle control incombination with 250 μg of anti-PD-1 antibody (-◯-), 5,000 ng ofCompound No. 63-10 in 95% sesame oil/5% ethanol (v/v) in combinationwith 250 μg of anti-PD-1 antibody (-▪-), 5,000 ng of Compound No. 63-33in 95% sesame oil/5% ethanol (v/v) in combination with 250 μg ofanti-PD-1 antibody (-▴-), or 5,000 ng of Compound No. 63-00 in phosphatebuffered saline in combination with 250 μg of anti-PD-1 antibody (-▾-)as described in Example B6. For all the treatment groups with ananti-PD-1 combination, the anti-PD-1 treatment was administeredintraperitoneally on experimental days 12, 15, 19, 22, and 26. Groupsize=10 for controls and all experimental conditions, the data isexpressed as average tumor volume (in mm³) +/− standard error of themean. Differences in tumor volumes between groups on experimental day 29were analyzed using a Kruskall-Wallis test followed by Dunn's post-testfor specific group pair comparisons. ns indicates P≥0.050; * indicatesP≤0.050; ** P≤0.010.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to alkyl chain modified1H-imidazo[4,5-c]quinoline derivatives that are potent TLR7/8 agonistsexhibiting balanced bioactivity against both receptors, and possessphysiochemical properties that enable pharmaceutical compositions whichpromote retention of the compound at the site of injection. The presentdisclosure also relates to pharmaceutical compositions comprising thealkyl chain modified 1H-imidazo[4,5-c]quinoline compounds and methods ofpreparation thereof, uses of the pharmaceutical compositions forstimulating an immune response, and to methods for the treatment ofdisease in a subject, e.g., infectious disease and cancer.

I. GENERAL METHODS AND DEFINITIONS

The practice of the present disclosure will employ, unless otherwiseindicated, conventional techniques of organic chemistry, analyticalchemistry, molecular biology, microbiology, cell biology, biochemistry,and immunology, which are within the skill of the art. Such techniquesare fully described in the literature, see for example: Fiesers'Reagents for Organic Synthesis, 25^(th) edition (Ho, ed., Wiley, 2016);Comprehensive Organic Functional Group Transformations, 2^(nd) edition(Katritsky and Taylor, eds., Elsevier, 2004); Comprehensive OrganicSynthesis, version 1-8 (Trost and Flemming, eds., Permagon Press, 1991);Beilsteins Handbuch der Organischen Chemie, 4 (Auflage, ed.,Springer-Verlag, 1934); Animal Cell Culture, sixth edition (Freshney,Wiley-Blackwell, 2010); Current Protocols in Cell Biology (Bonifacino etal., ed., John Wiley & Sons, Inc., 1996, including supplements through2014); Current Protocols in Immunology (Coligan et al., eds., John Wiley& Sons, Inc., 1991 including supplements through 2014); CurrentProtocols in Molecular Biology (Ausubel et al., eds., John Wiley & Sons,Inc., 1987, including supplements through 2014); Molecular Cloning: ALaboratory Manual, third edition (Sambrook and Russell, Cold SpringHarbor Laboratory Press, 2001); and Molecular Cloning: A LaboratoryManual, fourth edition (Green and Sambrook, Cold Spring HarborLaboratory Press, 2012).

The terms “individual” and “subject” refer to mammals. “Mammals”include, but are not limited to, humans, non-human primates (e.g.,monkeys), farm animals, sport animals (e.g., horses), rodents (e.g.,mice and rats), and pets (e.g., dogs and cats).

The term “antigen” refers to a substance that is recognized and boundspecifically by an antibody or by a T cell antigen receptor. Antigenscan include peptides, polypeptides, proteins, glycoproteins,polysaccharides, complex carbohydrates, sugars, gangliosides, lipids andphospholipids; portions thereof, and combinations thereof. Antigens whenpresent in the compositions of the present disclosure can be syntheticor isolated from nature. Antigens suitable for administration in themethods of the present disclosure include any molecule capable ofeliciting an antigen-specific B cell or T cell response. Haptens areincluded within the scope of “antigen.” A “hapten” is a low molecularweight compound that is not immunogenic by itself but is renderedimmunogenic when conjugated with a generally larger immunogenicmolecule.

“Polypeptide antigens” can include purified native peptides, syntheticpeptides, engineered peptides, recombinant peptides, crude peptideextracts, or peptides in a partially purified or unpurified active state(such as peptides that are part of attenuated or inactivated viruses,microorganisms or cells), or fragments of such peptides. Polypeptideantigens are preferably at least six amino acid residues in length,preferably from 8 to 1800 amino acids in length, more preferably from 9to 1000 amino acids in length, or from 10 to 100 amino acids in length.Similarly, in some embodiments, the polypeptide is about 9 to about2000, about 9 to about 1000, about 9 to about 100, or about 9 to about60 amino acids in length. In some embodiments, the polypeptide is atleast (lower limit) 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 60, 70, 80, or 90 amino acids in length. In someembodiments, the polypeptide is at most (upper limit) 1000, 900, 800,700, 600, 500, 400, 300, 250, 200, 150, 100, 50, or 25 amino acids inlength. In some embodiments, the polypeptide antigen is from 9 to 35amino acids in length.

As used herein, the term “immunogenic” refers to an agent (e.g.,polypeptide antigen) that elicits an adaptive immune response uponadministration under suitable conditions to a mammalian subject. Theimmune response may be a B cell (humoral) and/or T cell (cellular)mediated response.

“Adjuvant” refers to a substance which, when mixed with an immunogenicagent such as an antigen, nonspecifically enhances or potentiates animmune response to the agent in the recipient upon exposure to themixture.

The term “agonist” is used in the broadest sense and includes anymolecule that activates signaling through a receptor. For instance, aTLR7 agonist binds a toll-like receptor 7 protein and activates aTLR7-signaling pathway; a TLR8 agonist binds a toll-like receptor 8protein and activates a TLR8-signaling pathway. A dual TLR7/8 agonistbinds to both toll-like receptor 7 and toll-like receptor 8 proteins andactivates both TLR7- and TLR8-signaling pathways.

“Stimulation” of a response or parameter includes eliciting and/orenhancing that response or parameter when compared to conditions thatare otherwise the same except for the agent or molecule, oralternatively, as compared to another condition (e.g., increase inTLR-signaling in the presence of a TLR agonist as compared to theabsence of the TLR agonist). For example, “stimulation” of an immuneresponse means an increase in the response.

An “effective amount” of an agent disclosed herein is an amountsufficient to carry out a specifically stated purpose. An “effectiveamount” may be determined empirically and in a routine manner, inrelation to the stated purpose. An “effective amount” or an “amountsufficient” of an agent is that amount adequate to produce a desiredbiological effect, such as a beneficial result, including a beneficialclinical result. The term “therapeutically effective amount” refers toan amount of an agent (e.g., TLR modulator) effective to “treat” adisease or disorder in a subject (e.g., a mammal such as a human).

The terms “treating” or “treatment” of a disease refer to executing aprotocol, which may include administering one or more drugs to anindividual (human or otherwise), in an effort to alleviate signs orsymptoms of the disease. Thus, “treating” or “treatment” does notrequire complete alleviation of signs or symptoms, does not require acure, and specifically includes protocols that have only a palliativeeffect on the individual. As used herein, and as well-understood in theart, “treatment” is an approach for obtaining beneficial or desiredresults, including clinical results. Beneficial or desired clinicalresults include, but are not limited to, alleviation or amelioration ofone or more symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, preventing spread of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival of an individual not receivingtreatment.

“Palliating” a disease or disorder means that the extent and/orundesirable clinical manifestations of the disease or disorder arelessened and/or time course of progression of the disease or disorder isslowed, as compared to the expected untreated outcome. Especially in theallergy context, palliation may occur upon stimulation of a Th1 immuneresponse against an allergen(s). Further, palliation does notnecessarily occur by administration of one dose, but often occurs uponadministration of a series of doses. Thus, an amount sufficient topalliate a response or disorder may be administered in one or moredoses.

“Alkyl” as used herein refers to a saturated linear (i.e. unbranched) orbranched univalent hydrocarbon chain or combination thereof. Particularalkyl groups are those having a designated number of carbon atoms, forexample, an alkyl group having 1 to 20 carbon atoms (a “C₁-C₂₀ alkyl”),having 1 to 10 carbon atoms (a “C₁-C₁₀” alkyl), having 1 to 8 carbonatoms (a “C₁-C₈ alkyl”), having 1 to 6 carbon atoms (a “C₁-C₆ alkyl”),having 2 to 6 carbon atoms (a “C₂-C₆ alkyl”), or having 1 to 4 carbonatoms (a “C₁-C₄ alkyl”). Examples of alkyl groups include, but are notlimited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, isobutyl, sec-butyl, homologs and isomers of, for example,n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

“Alkenyl” as used herein refers to an unsaturated linear (i.e.unbranched) or branched univalent hydrocarbon chain or combinationthereof, having at least one site of olefinic unsaturation (i.e., havingat least one moiety of the formula C═C). Particular alkenyl groups arethose having a designated number of carbon atoms, for example, analkenyl group having 2 to 20 carbon atoms (a “C₂-C₂₀ alkenyl”), having 2to 10 carbon atoms (a “C₂-C₁₀” alkenyl), having 2 to 8 carbon atoms (a“C₂-C₈ alkenyl”), having 2 to 6 carbon atoms (a “C₂-C₆ alkenyl”), orhaving 2 to 4 carbon atoms (a “C₂-C₄ alkenyl”). The alkenyl group may bein “cis” or “trans” configurations, or alternatively in “E” or “Z”configurations. Examples of alkenyl groups include, but are not limitedto, groups such as ethenyl (or vinyl), prop-1-enyl, prop-2-enyl (orallyl), 2-methylprop-1-enyl, but-1-enyl, but-2-enyl, but-3-enyl,buta-1,3-dienyl, 2-methylbuta-1,3-dienyl, homologs, and isomers thereof,and the like.

“Alkynyl” as used herein refers to an unsaturated linear (i.e.unbranched) or branched univalent hydrocarbon chain or combinationthereof, having at least one site of acetylenic unsaturation (i.e.,having at least one moiety of the formula C≡C). Particular alkynylgroups are those having a designated number of carbon atoms, forexample, an alkynyl group having 2 to 20 carbon atoms (a “C₂-C₂₀alkynyl”), having 2 to 10 carbon atoms (a “C₂-C₁₀ alkynyl”), having 2 to8 carbon atoms (a “C₂-C₈ alkynyl”), having 2 to 6 carbon atoms (a “C₂-C₆alkynyl”), or having 2 to 4 carbon atoms (a “C₂-C₄ alkynyl”). Examplesof alkynyl groups include, but are not limited to, groups such asethynyl (or acetylenyl), prop-1-ynyl, prop-2-ynyl (or propargyl),but-1-ynyl, but-2-ynyl, but-3-ynyl, homologs, and isomers thereof, andthe like.

“Alkylene” as used herein refers to the same residues as alkyl, buthaving bivalency. Particular alkylene groups are those having 1 to 6carbon atoms (a “C₁-C₆ alkylene”), 1 to 5 carbon atoms (a “C₁-C₅alkylene”), 1 to 4 carbon atoms (a “C₁-C₄ alkylene”), or 1 to 3 carbonatoms (a “C₁-C₃ alkylene”). Examples of alkylene groups include, but arenot limited to, groups such as methylene (—CH₂— or ═CH₂), ethylene(—CH₂CH₂— or ═CHCH₃), propylene (—CH₂CH₂CH₂— or ═CHCH₂CH₃), butylene(—CH₂CH₂CH₂CH₂— or ═CHCH₂CH₂CH₃), and the like.

“Cycloalkyl” as used herein refers to non-aromatic, saturated, orunsaturated cyclic univalent hydrocarbon structures. Particularcycloalkyl groups are those having a designated number of annular (i.e.,ring) carbon atoms, for example, a cycloalkyl group having from 3 to 12annular carbon atoms (a “C₃-C₁₂ cycloalkyl”). A preferred cycloalkyl isa cyclic hydrocarbon having from 3 to 8 annular carbon atoms (a “C₃-C₈cycloalkyl”), or having 3 to 6 annular carbon atoms (a “C₃-C₆cycloalkyl”). Cycloalkyl can consist of one ring, such as cyclohexyl, ormultiple rings, such as adamantyl, but excludes aryl groups. Acycloalkyl comprising more than one ring may be fused, spiro, orbridged, or combinations thereof. Examples of cycloalkyl groups include,but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, norbornyl, andthe like.

“Cycloalkylene” as used herein refers to the same residues ascycloalkyl, but having bivalency. Particular cycloalkylene groups arethose having 3 to 12 annular carbon atoms (a “C₃-C₁₂ cycloalkylene”),having from 3 to 8 annular carbon atoms (a “C₃-C₈ cycloalkylene”), orhaving 3 to 6 annular carbon atoms (a “C₃-C₆ cycloalkylene”). Examplesof cycloalkylene groups include, but are not limited to, cyclopropylene,cyclobutylene, cyclopentylene, cyclohexylene, 1,2-cyclohexenylene,1,3-cyclohexenylene, 1,4-cyclohexenylene, cycloheptyl, norbornyl, andthe like. “Hydrocarbyl” as used herein refers to and includes aunivalent group formed by removing a hydrogen atom from a non-aromatichydrocarbon, which may be fully saturated mono- or polyunsaturated,having the number of carbon atoms designated (i.e., C₁-C₂₀ means one totwenty carbon atoms). A hydrocarbyl group may contain one or morelinear, branched, or cyclic moieties, or combinations thereof. Alkyl,alkenyl, alkynyl, and cycloalkyl groups are particular subsets ofhydrocarbyl groups. A hydrocarbyl group may also contain an alkyl,alkenyl or alkynyl group further substituted by one or more cycloalkylgroups; and/or a cycloalkyl group further substituted by one of morealkyl, alkenyl, and/or alkynyl groups. A hydrocarbyl group may besubstituted, at one or more positions, by one or more halogen atoms,such as chlorine or fluorine. Examples of hydrocarbyl groups include,but are not limited to, groups such as the following:

and the like.

“Aryl” as used herein refers to an unsaturated aromatic carbocyclicgroup having a single ring (e.g., phenyl) or multiple condensed rings(e.g., naphthyl or anthryl), where one or more of the condensed ringsmay not be aromatic. Particular aryl groups are those having from 6 to14 annular (i.e., ring) carbon atoms (a “C₆-C₁₄ aryl”). An aryl grouphaving more than one ring where at least one ring is non-aromatic may beconnected to the parent structure at either an aromatic ring position orat a non-aromatic ring position. In one variation, an aryl group havingmore than one ring where at least one ring is non-aromatic is connectedto the parent structure at an aromatic ring position. Examples of arylinclude, but are not limited to, groups such as phenyl, naphthyl,1-naphthyl, 2-naphthyl, and the like.

“Arylene” as used herein refers to the same residues as aryl, but havingbivalency. Particular arylene groups are those having from 6 to 14annular carbon atoms (a “C₆-C₁₄ arylene”). Examples of arylene include,but are not limited to, groups such as phenylene, o-phenylene (i.e.,1,2-phenylene), m-phenylene (i.e., 1,3-phenylene), p-phenylene (i.e.,1,4-phenylene), naphthylene, 1,2-naphthylene, 1,2-naphthylene,1,4-naphthylene, and the like.

“Halo” or “halogen” refers to elements of the Group 17 series havingatomic number 9 to 85. Preferred halo groups include fluoro, chloro,bromo, and iodo. Where a residue is substituted with more than onehalogen, it may be referred to by using a prefix corresponding to thenumber of halogen moieties attached. For example, dihaloaryl,dihaloalkyl, and trihaloaryl etc., refer to aryl and alkyl substitutedwith two (“di”) or three (“tri”) halo groups, which may be but are notnecessarily the same halo; thus 4-chloro-3-fluorophenyl is within thescope of dihaloaryl. An alkyl group in which each hydrogen is replacedwith a halo group is referred to as a “perhaloalkyl.” A preferredperhaloalkyl group is trifluoroalkyl (—CF₃). Similarly, “perhaloalkoxy”refers to an alkoxy group in which a halogen takes the place of each Hin the hydrocarbon making up the alkyl moiety of the alkoxy group. Anexample of a perhaloalkoxy group is trifluoromethoxy (—OCF₃).

“Amino” refers to the group —NH₂.

“Substituted amino” refers to the group —NR′R″ where R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,substituted alkynyl, aryl, heteroaryl, and heterocyclyl provided that atleast one of R′ and R″ is not hydrogen.

“Optionally substituted” unless otherwise specified means that a groupmay be unsubstituted or substituted by one or more (e.g., 1, 2, 3, 4, or5) of the substituents listed for that group in which the sub stituentsmay be the same or different. In one embodiment, an optionallysubstituted group has one substituent. In another embodiment, anoptionally substituted group has two substituents. In anotherembodiment, an optionally substituted group has three substituents. Inanother embodiment, an optionally substituted group has foursubstituents. In some embodiments, an optionally substituted group has 1to 2, 1 to 3, 1 to 4, or 1 to 5 substituents.

“Organic modifier” unless otherwise specified means one of a group ofsolvents typically used to solubilize organic chemical compounds. Thisgroup can include, but is not limited to acetic acid, acetone, anisole,1-butanol, 2-butanol, butyl acetate, tert-butyl methyl ether, cumene,dimethyl sulfoxide, ethanol, ethyl acetate, ethyl ether, ethyl formate,formic acid, heptane, isobutyl acetate, isopropyl acetate, methylacetate, 3-methyl-1-butanol, methylenthylketone, methylisobutylketone,2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol(isopropanol), propyl acetate, and combinations thereof.

In addition to the disclosure herein, the term “substituted,” when usedto modify a specified group or radical, can also mean that one or morehydrogen atoms of the specified group or radical are each, independentlyof one another, replaced with the same or different substituent groupsas defined herein. In some embodiments, a group that is substituted has1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2substituents, or 1 substituent.

Unless a specific isotope of an element is indicated in a formula, theinvention includes all isotopologues of the compounds disclosed herein,such as, for example, deuterated derivatives of the compounds (where Hcan be ²H, i.e., D). Isotopologues can have isotopic replacements at anyor at all locations in a structure, or can have atoms present in naturalabundance at any or all locations in a structure.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination. All combinations of the embodimentspertaining to the chemical groups represented by the variables arespecifically embraced by the present invention and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed, to the extent that such combinations embrace compounds thatare stable compounds (i.e., compounds that can be isolated,characterized, and tested for biological activity). In addition, allsubcombinations of the chemical groups listed in the embodimentsdescribing such variables are also specifically embraced by the presentinvention and are disclosed herein just as if each and every suchsub-combination of chemical groups was individually and explicitlydisclosed herein.

It is understood that aspects and embodiments described herein as“comprising” include “consisting of” and “consisting essentially of”embodiments.

As used herein and in the appended claims, the singular forms “a,” “an”,and “the” include plural referents unless otherwise indicated or clearfrom context.

Unless clearly indicated otherwise, the term “about” is used to indicatethat a value includes the standard deviation or error for the device ormethod being employed to determine the value. Reference to “about” avalue or parameter herein includes (and describes) embodiments that aredirected to that value or parameter per se. For example, descriptionreferring to “about X” includes description of “X.”

II. COMPOUNDS

In one aspect, provided is a compound of formula (J):

or a salt thereof, wherein:

-   -   R⁰ is C₄-C₂₁ hydrocarbyl optionally substituted by 1 to 4        halogen atoms;    -   X is —NH— or —NH(C═O)—;    -   R¹ is C₃-C₆ alkyl, —(CH₂)_(p)OR^(1a), —(CH₂)_(p)NHR^(1b), or        —(CH₂)_(p)R^(1c); where R^(1a) and R^(1b) are independently        C₁-C₃ alkyl; R^(1c) is C₃-C₄ cycloalkyl; and p is 1 or 2;    -   R² is NHR^(2a); where R^(2a) is H, OH, NH₂, or methyl;    -   each R³ is independently halogen, C₁-C₈ alkyl, —(C₁-C₇        alkylene)—NH₂, or    -   —CH₂-phenylene-CH₂NH₂;    -   q is 0, 1, 2, 3, or 4; and    -   R^(4a) and R^(4b) are independently H or C₁-C₈ alkyl,    -   provided that the compound is other than        2-butyl-1-(4-((hexadecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine        (Compound No. 63-32);        N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)palmitamide        (Compound No. 63-31); or        N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)pent-4-ynamide        (Compound No. 63-37).

In some embodiments, R⁰ is C₄-C₂₁ hydrocarbyl. In some embodiments, R⁰is C₄-C₁₄ hydrocarbyl. In some embodiments, R⁰ is C₅-C₁₀ hydrocarbyl. Insome embodiments, R⁰ is C₁₀-C₁₄ hydrocarbyl. In some embodiments, R⁰ isC₅-C₇ hydrocarbyl.

In some embodiments, X is —NH(C═O)—. In other embodiments, X is —NH—.

In some embodiments, R⁰ is branched C₄-C₁₄ alkyl or —(CH₂)_(m)R^(A); mis 0, 1, 2, or 3; and R^(A) is C₃-C₈ cycloalkyl optionally substitutedby 1 to 4 groups independently selected from the group consisting ofC₁-C₄ alkyl and C₁-C₄ alkylene.

In some embodiments, R⁰ is branched C₄-C₁₄ alkyl. In some embodiments,R⁰ is branched C₅-C₁₀ alkyl. In some embodiments, R⁰ is branched C₁₀-C₁₄alkyl. In some embodiments, R⁰ is branched C₅-C₇ alkyl.

In some embodiments, R⁰ is —(CH₂)_(m)R^(A). In one variation, m is 1 or2, and R^(A) is cyclopropyl, cyclobutyl, or cyclopentyl. In anothervariation, m is 0, and R^(A) is cyclobutyl, cyclopentyl, or cyclohexyl.

In some embodiments, R^(A) is C₃-C₈ cycloalkyl.

In some embodiments, R^(A) is C₃-C₆ cycloalkyl optionally substituted by1 to 3 groups independently selected from the group consisting of methyland methylene. In one variation, m is 1 or 2.

In some embodiments, R^(A) is cyclopropyl optionally substituted by 1 to3 groups independently selected from the group consisting of methyl andmethylene, and m is 1 or 2.

In some embodiments, m is 0 or 1, and R^(A) is cyclohexyl optionallysubstituted by 1 to 3 groups independently selected from the groupconsisting of methyl and methylene.

In some embodiments, R^(A) is C₃-C₆ cycloalkyl optionally substituted by1 to 4 halogen atoms. In some embodiments, R^(A) is C₃-C₆ cycloalkyloptionally substituted by 1 to 3 chlorine or fluorine atoms. In someembodiments, R^(A) is C₃-C₆ cycloalkyl optionally substituted by 1 to 2chlorine or fluorine atoms. In some embodiments, R^(A) is cyclobutyloptionally substituted by 1 to 2 fluorine atoms. In one variation, m is1.

In some embodiments, R⁰ is —(CH₂)_(z)(C(CH₃)₂)R^(A). In one variation, zis 1 or 2, and R^(A) is cyclopropyl, cyclobutyl, or cyclopentyl. In onevariation, z is 1, and R^(A) is cyclopropyl, cyclobutyl, or cyclopentyl.

In some embodiments, R⁰ is selected from the group consisting of:

In some embodiments, R⁰ is selected from the group consisting of:

In some embodiments, R⁰ is selected from the group consisting of:

In some embodiments, X is —NH—, R⁰ is —(CH₂)_(m)R^(A), m is 2, and R^(A)is cyclopropyl, cyclobutyl, or cyclopentyl.

In some embodiments, X is —NH—, R⁰ is —(CH₂)_(z)(C(CH₃)₂)R^(A), z is 1,and R^(A) is cyclopropyl, cyclobutyl, or cyclopentyl.

In some embodiments, X is —NH—, R⁰ is —(CH₂)_(m)R^(A), m is 0, and R^(A)is cyclobutyl, cyclopentyl, or cyclohexyl.

In some embodiments, X is —NH(C═O)—, R⁰ is —(CH₂)_(m)R^(A), m is 1, andR^(A) is cyclopropyl, cyclobutyl, or cyclopentyl.

In some embodiments, R¹ is C₃-C₆ alkyl (e.g., n-butyl). In someembodiments, R¹ is propyl, butyl, pentyl, or hexyl. In some preferredembodiments, R¹ is n-butyl. In some embodiments, R¹ is n-pentyl. In someembodiments, R¹ is —(CH₂)_(p)OR^(1a) (e.g., CH₂OCH₂CH₃). In someembodiments, R¹ is —(CH₂)_(p)NHR^(1b) (e.g., CH₂NHCH₂CH₃). In someembodiments, R¹ is —(CH₂)_(p)R^(1c). In one variation, R^(1c) iscyclopropyl.

In some embodiments, R² is NHR^(2a), where R^(2a) is H, OH, NH₂, ormethyl. In some embodiments, R² is NH₂. In some embodiments, R² is NHOH,NHNH₂, or NHCH₃.

In some embodiments, R^(4a) and R^(4b) are independently H or C₁-C₈alkyl. In some embodiments, each R^(4a) and R^(4b) is H.

In some embodiments, the phenyl moiety of the 1H-imidazo[4,5-c]quinolinecore is unsubstituted (i.e., q is 0). In some embodiments, the phenylmoiety of the 1H-imidazo[4,5-c]quinoline core is substituted by 1, 2, 3,or 4 substituents independently selected from the group consisting ofhalogen, C₁-C₈ alkyl, —(C₁-C₇ alkylene)—NH₂, and —CH₂-phenylene-CH₂NH₂.In some embodiments, q is 1, and R³ is C₁-C₈ alkyl.

It is intended and understood that where present each and everyvariation of X and R⁰ described for formula (J) may be combined witheach and every variation of R¹, p, R², q, R³, R^(4a) and R^(4b)described for formula (J) the same as if each and every combination isspecifically and individually described.

In some embodiments, the compound of formula (J) is of formula (J-1):

or a salt thereof, wherein R⁰ is C₄-C₂₁ hydrocarbyl. In someembodiments, R⁰ is —(CH₂)_(m)R^(A). In one variation, m is 1 or 2, andR^(A) is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In onevariation, m is 2, and R^(A) is cyclopropyl, cyclobutyl, or cyclopentyl.In another variation, m is 0, and R^(A) is cyclobutyl, cyclopentyl, orcyclohexyl. In some embodiments, R⁰ is —(CH₂)_(z)(C(CH₃)₂)R^(A). In onevariation, z is 1, and R^(A) is cyclopropyl, cyclobutyl, or cyclopentyl.In some embodiments, R^(A) is optionally substituted by 1-4 groupsindependently selected from the group consisting of methyl, methylene,and halogen.

In some embodiments, the compound of formula (J) is of formula (J-2):

or a salt thereof, wherein R⁰ is C₄-C₂₁ hydrocarbyl, provided that thecompound is other thanN-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)pent-4-ynamide(Compound No. 63-37). In some embodiments, R⁰ is —(CH₂)_(m)R^(A). In onevariation, m is 1 or 2, and R^(A) is cyclopropyl, cyclobutyl,cyclopentyl, or cyclohexyl. In one variation, m is 1 and R^(A) iscyclopropyl.

In another aspect, provided is a compound of formula (K):

or a salt thereof, wherein:

n is an integer from 4 to 21;

X is —NH— or —NH(C═O)—;

-   -   R¹ is C₃-C₆ alkyl, —(CH₂)_(p)OR^(1a), —(CH₂)_(p)NH R^(1b) or        —(CH₂)_(p)R^(1c); where R^(1a) and R^(1b) are independently        C₁-C₃ alkyl; R^(1c) is C₃-C₄ cycloalkyl; and p is 1 or 2;

R² is NHR^(2a); where R^(2a) is H, OH, NH₂ or methyl;

each R³ is independently halogen, C₁-C₈ alkyl, —(C₁-C₇ alkylene)—NH₂, or—CH₂-phenylene-CH₂NH₂;

q is 0, 1, 2, 3, or 4; and

R^(4a) and R^(4b) are independently H or C₁-C₈ alkyl,

-   -   provided that the compound is other than        2-butyl-1-(4-((hexadecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine        (Compound No. 63-32) or        N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)palmitamide        (Compound No. 63-31).

In some embodiments, X is —NH—. In some embodiments, X is —NH(C═O)—.

In some embodiments, X is —NH— and n is an integer from 4 to 15. In somepreferred embodiments, X is —NH— and n is an integer from 4 to 12. Insome preferred embodiments, X is —NH— and n is 4, 5, 6, or 7. In someembodiments, n is 8, 9, 10, 11, 12, 13, 14, or 15.

In some embodiments, X is —NH(C═O)— and n is an integer from 4 to 14. Insome preferred embodiments, X is —NH(C═O)— and n is 11, 12, 13, or 14.In some embodiments, n is 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, R¹ is C₃-C₆ alkyl. In some embodiments, R¹ ispropyl, butyl, pentyl or hexyl. In some preferred embodiments, R¹ isn-butyl. In some embodiments, R¹ is n-pentyl.

In some embodiments, R¹ is —(CH₂)_(p)OR^(1a), where p is 1 or 2 andR^(1a) is C₁-C₃ alkyl. In some embodiments, R¹ is —CH₂OCH₂CH₃.

In some embodiments, R¹ is —(CH₂)_(p)NHR^(1b) where R^(1b) is C₁-C₃alkyl. In some embodiments, R¹ is —CH₂NHCH₂CH₃.

In some embodiments, R¹ is —(CH₂)_(p)R^(1c) where p is 1 or 2 and R^(1c)is cyclopropyl or cyclobutyl. In some embodiments, R¹ is—CH₂-cyclopropyl or —CH₂CH₂-cyclopropyl.

In some embodiments, R² is NHR^(2a), where R^(2a) is H, OH, NH₂, ormethyl. In some preferred embodiments, R² is NH₂. In some embodiments,R² is NHOH, NHNH₂, or NHCH₃.

In some embodiments, the phenyl moiety of the 1H-imidazo[4,5-c]quinolinecore is unsubstituted (i.e., q is 0). In some embodiments, the phenylmoiety of the 1H-imidazo[4,5-c]quinoline core is substituted by 1, 2, 3,or 4 substituents independently selected from the group consisting ofhalogen, C₁-C₈ alkyl, —(C₁-C₇ alkylene)—NH₂, and —CH₂-phenylene-CH₂NH₂.In some embodiments, q is 1 and R³ is C₁-C₈ alkyl.

In some embodiments, R^(4a) and R^(4b) are independently H or C₁-C₈alkyl. In some preferred embodiments, each R^(4a) and R^(4b) is H.

It is intended and understood that each and every variation of X and ndescribed for formula (K) may be combined with each and every variationof R¹, R², q, R³, R^(4a), and R^(4b), where present, described forformula (K) the same as if each and every combination is specificallyand individually described. For example, in some embodiments, R¹ isC₃-C₆ alkyl (e.g., n-butyl), R² is NH₂, q is 0, X is —NH—, and n is 4,5, 6, or 7. In some embodiments, R¹ is C₃-C₆ alkyl (e.g., n-butyl), R²is NH₂, q is 0, X is —NH(C═O)—, and n is 11, 12, 13, or 14.

In some embodiments, the compound of formula (K) is of formula (K-1):

or a salt thereof, wherein R″ is linear C₄-C₂₁ alkyl, provided that thecompound is other than2-butyl-1-(4-((hexadecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine(Compound No. 63-32). In some embodiments, R″ is linear C₄-C₁₅ alkyl. Insome embodiments, R″ is linear C₄-C₇ alkyl. In some embodiments, R″ islinear C₈-C₁₅ alkyl. In some embodiments, R″ is linear C₁₇-C₂₁ alkyl.

In some embodiments, the compound of formula (K) is of formula (K-2):

or a salt thereof, wherein R is linear C₄-C₂₁ alkyl, provided that thecompound is other thanN-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)palmitamide(Compound No. 63-31). In some embodiments, R is linear C₄-C₁₄ alkyl. Insome embodiments, R is linear C₁₁-C₁₄ alkyl. In some embodiments, R islinear C₄-C₁₀ alkyl. In some embodiments, R is linear C₁₆-C₂₁ alkyl.

Representative compounds of the invention are listed in Table 1.

TABLE 1 Compound No. Formula Name 63-01

N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)pentanamide 63-02

N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)hexanamide 63-03

N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)heptanamide 63-04

N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)octanamide 63-05

N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)nonanamide 63-06

N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)decanamide 63-07

N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)undecanamide 63-08

N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)dodecanamide 63-09

N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)tridecanamide 63-10

N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)tetradecanamide 63-11

N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)pentadecanamide 63-12

N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)heptadecanamide 63-13

N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)stearamide 63-14

N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)nonadecanamide 63-15

N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)icosanamide 63-16

2-butyl-1-(4-((butylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-17

2-butyl-1-(4-((pentylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-18

2-butyl-1-(4-((hexylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-19

2-butyl-1-(4-((heptylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-20

2-butyl-1-(4-((octylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-21

2-butyl-1-(4-((nonylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-22

2-butyl-1-(4-((decylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-23

2-butyl-1-(4-((undecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-24

2-butyl-1-(4-((dodecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-25

2-butyl-1-(4-((tridecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-26

2-butyl-1-(4-((tetradecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-27

2-butyl-1-(4-((pentadecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-28

2-butyl-1-(4-((heptadecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-29

2-butyl-1-(4-((octadecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-30

2-butyl-1-(4-((nonadecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-33

2-butyl-1-(4-(((2- cyclopropylethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-34

N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-2-cyclopropylacetamide 63-35

2-butyl-1-(4-(((2-cyclobutylethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-36

2-butyl-1-(4-(((2-cyclopentylethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-38

2-butyl-1-(4-(((cyclopropylmethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-39

2-butyl-1-(4-((((2- methylcyclopropyl)methyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinoline-4-amine 63-40

2-butyl-1-(4-((((2,2-dimethylcyclopropyl)methyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-41

2-butyl-1-(4-(((2-cyclopropyl-2-methylpropyl)amino)methyl)benzyl)-1H-imidazo[4,5- c]quinoline-4-amine63-42

2-butyl-1-(4-(((2-(1- methylcyclopropyl)ethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-43

2-butyl-1-(4-(((3- cyclopropylpropyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-44

2-butyl-1-(4-(((cyclobutylmethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-45

2-butyl-1-(4-((((1- methylcyclobutyl)methyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinoline-4-amine 63-46

2-butyl-1-(4-((((3- methylcyclobutyl)methyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-47

2-butyl-1-(4-(((2-cyclobutyl-2-methylpropyl)amino)methyl)benzyl)-1H-imidazo[4,5- c]quinolin-4-amine63-48

2-butyl-1-(4-((((3- fluorocyclobutyl)methyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinoline-4-amine 63-49

2-butyl-1-(4-((cyclohexylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine

Additional compounds are listed in Table 2.

TABLE 2 Compound No. Formula Name 63-00

1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5- c]quinolin-4-amine63-31

N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)palmitamide 63-32

2-butyl-1-(4-((hexadecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine 63-37

N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)pent-4-ynamide

In some embodiments, the compounds of the invention exclude those listedin Table 2.

In some embodiments, provided is a compound selected from Compounds Nos.63-01 to 63-30, 63-33 to 63-36, and 63-38 to 63-49 in Table 1, or a saltthereof. In some embodiments, the compound is selected from the groupconsisting of one or more of Compound Nos. 63-01 to 63-30 in Table 1, ora salt thereof. In some embodiments, the compound is selected from thegroup consisting of one or more of Compound Nos. 63-33 to 63-36 and63-38 to 63-49 in Table 1, or a salt thereof.

The invention also includes all salts of compounds referred to herein,such as pharmaceutically acceptable salts. The invention also includesany or all of the stereochemical forms, including any enantiomeric ordiastereomeric forms, and any tautomers or other forms of the compoundsdescribed. Unless stereochemistry is explicitly indicated in a chemicalstructure or name, the structure or name is intended to embrace allpossible stereoisomers of a compound depicted. In addition, where aspecific stereochemical form is depicted, it is understood that otherstereochemical forms are also embraced by the invention. All forms ofthe compounds are also embraced by the invention, such as crystalline ornon-crystalline forms of the compounds. Compositions comprising acompound of the invention are also intended, such as a composition ofsubstantially pure compound, including a specific stereochemical formthereof. Compositions comprising a mixture of compounds of the inventionin any ratio are also embraced by the invention, including mixtures oftwo or more stereochemical forms of a compound of the invention in anyratio, such that racemic, non-racemic, enantioenriched and scalemicmixtures of a compound are embraced.

Compounds of the present disclosure are potent TLR7/8 agonistsexhibiting balanced bioactivity against both receptors and possessphysiochemical properties such as increased hydrophobicity. Compoundswith potent agonist bioactivity against both TLR7 and TLR8 receptors arepotentially more effective immune adjuvants than agonists specific foronly one of these TLRs, and would promote innate immune responses in awider range of antigen presenting cells and other key immune cell types,including plasmacytoid and myeloid dendritic cells, monocytes, and Bcells (see e.g., Vasilakos et al 2013 Expert Rev Vaccines 12:809-819).Compounds with physiochemical properties such as increasedhydrophobicity are known to be compatible with oil-based formulationapproaches, and enable pharmaceutical compositions that promoteretention of the compound at the site of injection.

In some embodiments, compounds of formula (J) or (K) are capable ofactivating both TLR7 and TLR8. In some embodiments, the compound offormula (J) or (K) has an EC₅₀ for TLR7 of about 200 nM or lower and anEC₅₀ for TLR8 of about 2000 nM or lower, wherein the EC₅₀ values are asdescribed in Example B 1. In some embodiments, the compound of formula(J) or (K) has an EC₅₀ for TLR7 of about 50 nM or lower, and an EC₅₀ forTLR8 of about 1000 nM or lower. In some embodiments, the compound offormula (J) or (K) has an EC₅₀ for TLR7 of about 200 nM, about 175 nM,about 150 nM, about 125 nM, about 100 nM, about 75 nM, about 50 nM,about 40 nM, about 30 nM, about 20 nM, about 10 nM, about 8 nM, about 6nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, or about0.5 nM; and an EC₅₀ for TLR8 of about 2000 nM, 1500 nM, 1000 nM, 900 nM,800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 50 nM,25 nM, 10 nM, 5 nM, 1 nM, or 0.5 nM.

Compounds of formula (J-1) or (K-1) are N-alkyl derivatives of1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine(IMDQ); while compounds of formula (J-2) or (K-2) are N-acyl derivativesof IMDQ. IMDQ (Compound No. 63-00) shows potent in vitro activities forboth TLR7 and TLR8 receptors (see e.g., U.S. Pat. Nos. 8,728,486 and9,441,005). Alkyl chain modification of compounds of the presentinvention at the benzylic amine increases hydrophobicity, and therebyenables pharmaceutical compositions that promote retention of thecompound at the site of injection. However, while alkyl chainderivatives of increasing carbon chain length may provide significantincreases in hydrophobicity (for example as estimated by the calculatedpartitioning coefficient or cLogP), these derivatives can also havediminished agonist potency against both TLR7 and TLR8, or selectivelyhave diminished bioactivity against one of the two receptors. Forexample, the N-octadecanoyl derivative (Compound No. 63-13) is over40-fold less potent against TLR7 and 26-fold less potent against TLR8than its parent congener IMDQ in the same in vitro human immune cellbioactivity assays. Unexpectedly, the N-tetradecanoyl derivative(Compound No. 63-10) of IMDQ is only 2 and 2.4-fold less potent againstTLR7 and TLR8, respectively, than IMDQ. These data demonstrate that analkyl chain of optimal length provides potent balanced TLR7/8 agonistbioactivity, as well as increased hydrophobicity that allows forincorporation into pharmaceutical compositions that promote retention ofthe compound at the site of injection.

Compounds of formula (J-1) or (K-1) are N-alkyl derivatives of1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine(IMDQ). While alkyl chain derivatives of increasing carbon chain lengthmay provide increases in hydrophobicity (for example as estimated by thecalculated partitioning coefficient or cLogP), these derivatives canalso have diminished agonist potency against both TLR7 and TLR8, orselectively have diminished bioactivity against one of the tworeceptors. For example, the 4-octadecylamino derivative (Compound No.63-29) is 19-fold less potent against TLR7 and over 12-fold less potentagainst TLR8 than its parent congener IMDQ in the same in vitro humanimmune cell bioactivity assays (see e.g., Example B1). Interestingly,the 4-pentylamino derivative (Compound No. 63-17) is only 2-fold lesspotent against TLR7 and is 2-fold more potent against TLR8 than IMDQ,but shows significantly less hydrophobicity than Compound No. 63-29.Unexpectedly, the 4-(2-cyclopropylethyl)amino derivative (Compound No.63-33) is only 4-fold less potent against TLR7 and 2.9-fold more potentagainst TLR8 than IMDQ, and has an increased cLogP compared to thelinear 5 carbon variant (Compound No. 63-17). These data demonstratethat an alkyl chain of optimal length provides potent balanced TLR7/8agonist bioactivity, as well as increased hydrophobicity that may allowfor incorporation into pharmaceutical compositions that promoteretention of the compound at the site of injection.

A TLR7/8 agonist small molecule with balanced dual potency would alsoallow for the synthesis and characterization of a single activepharmaceutical ingredient, thereby facilitating GMP manufacturing atlower costs and enabling a more straightforward and predictableregulatory pathway.

Compounds of formula (J) may be synthesized according to Scheme 1 and/orusing methods known in the art.

wherein R¹, R², q, and R³ are as defined for formula (J), R, and R⁰ arehydrocarbyl groups.

Preferred compounds of formula (J) where X is —NH—, R⁰ is—(CH₂)_(m)R^(A), m is 1 or 2, and R^(A) is cyclopropyl, cyclobutyl,cyclopentyl, or cyclohexyl can be prepared using the followingcarboxylic acids: cyclopropanecarboxylic acid, cyclopropyl acetic acid,cyclobutanecarboxylic acid, cyclobutylacetic acid,cyclopentanecarboxylic acid, cyclopentylacetic acid,cyclohexanecarboxylic acid, and cyclohexaneacetic acid. Preferredcompounds of formula (J) where X is —NH—, R⁰ is —(CH₂)_(m)R^(A), m is 1,2, or 3, and R^(A) is cyclopropyl, 2-methylcyclopropyl,2,2,-dimethylcyclopropyl, 1-methylcyclopropyl, 1-methylcyclobutyl,3-methylcyclobutyl, or 3-fluorocyclobutyl can be prepared using thefollowing carboxylic acids: cyclopropanecarboxylic acid,2-methylcyclopropane carboxylic acid, 2,2-dimethylcyclopropanecarboxylic acid, 1-methylcyclopropane carboxylic acid,1-methylcyclobutane carboxylic acid, 3-methylcyclobutane carboxylicacid, or 3-fluorocyclobutane carboxylic acid. Preferred compounds offormula (J) where X is —NH—, R⁰ is —(CH₂)(C(CH₃)₂)R^(A), and R^(A) iscyclopropyl or cyclobutyl can be prepared using the following carboxylicacids: cyclopropane carboxylic acid or cyclobutane carboxylic acid. Adetailed description of the synthesis scheme for the representativeCompound No. 63-33 can be found in Example S3.

In some embodiments, where R¹ is C₃-C₆ alkyl (e.g., n-butyl), R² is NH₂,X is —NH—, R⁰ is —(CH₂)_(m)R^(A), m is 0, and R^(A) is cycloalkyl, thecompounds are synthesized according to Scheme 1-2. A compound of formula(J), wherein R¹ is n-butyl, R² is NH₂, X is —NH—, R⁰ is —(CH₂)_(m)R^(A),m is 0, and R^(A) is cyclohexyl (Compound No. 63-49) is preparedaccording to the synthesis described in Example S10.

wherein R¹, q, and R³ are as defined for formula (J), and R is acycloalkyl group.

Compounds of formula (K) may be synthesized according to Scheme 2 and/orusing methods known in the art.

wherein R¹, R², q, and R³ are as defined for formula (K), R, and R″ arelinear alkyl groups.

Preferred compounds of formula (K) where X is —NH(C═O)— and n is aninteger from 4 to 21 provided that the compound is other thanN-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)palmitamide(Compound No. 63-31) can be prepared using the following carboxylicacids; pentanoic, hexanoic, heptaonic, octanoic, nonanoic, decanoic,undecanoic, dodecanoic, tridecanoic, tetradecanoic, pentadecanoic,hexadecanoic, heptadecanoic, octadecanoic, nonadecanoic, icosanoic,heneicosanoic, and docosanoic. A detailed description of the synthesisscheme for the representative Compound Nos. 63-10 and 63-17 can be foundin Examples S1 and S2, respectively.

In some embodiments, where R¹ is C₃-C₆ alkyl (e.g., n-butyl), R² is NH₂,and q is 0, the compounds are synthesized according to Scheme 3 or 4.For more detailed description of the individual reaction steps usefulfor preparing Compound No. 63-00, the starting compound in Schemes 3 and4, see e.g., U.S. Pat. Nos. 8,728,486 and 9,441,005.

wherein R and R⁰ are hydrocarbyl groups.

wherein R and R″ are linear alkyl groups.

Those skilled in the art will appreciate that other synthetic routes maybe employed to synthesize the range of compounds described within theinvention including various solvents, catalysts, reducing agents,temperatures, reaction times, and atmospheric conditions.

Conventional methods and techniques of separation and purification canbe used to isolate the compounds of the invention. Techniques mayinclude high performance liquid chromatography (HPLC) with differentmatrices (see e.g., C18, C8, C4, etc.), chromatography using typicaladsorbants (see e.g., silica gel, activated carbon, alumina, zeolites,and the like), recrystallization, and differential extraction methods(e.g., liquid-liquid, solid phase, and the like).

III. PHARMACEUTICAL COMPOSITIONS

Pharmaceutical compositions comprising alkyl chain modified1H-imidazo[4,5-c]quinoline TLR7/8 agonists of the present disclosure arealso provided. The pharmaceutical compositions routinely contain one ormore pharmaceutically acceptable excipients. In some embodiments, thepharmaceutical compositions further comprise an antigen. Thepharmaceutical compositions of the present disclosure are preferablysterile, and preferably essentially endotoxin-free.

Excipients

Pharmaceutically acceptable excipients of the present disclosureinclude, for instance, oils, lipids, solvents, bulking agents,surfactants, buffering agents, tonicity adjusting agents, andpreservatives (see, e.g., Pramanick et al 2013 Pharma Times 45:65-77).In some embodiments, the pharmaceutical compositions comprise anexcipient that functions as one or more of a solvent, a bulking agent, abuffering agent, and a tonicity adjusting agent (e.g., sodium chloridein saline may serve as both an aqueous vehicle and a tonicity adjustingagent). The pharmaceutical compositions of the present disclosure aresuitable for parenteral routes of administration, and in certaininstances preferentially by intratumoral administration. In certainembodiments, the pharmaceutical compositions of the present disclosureare not intended for enteral administration.

In some embodiments, the pharmaceutical compositions comprise anoil-based excipient to solubilize the TLR7/8 agonist compound so as toenable parenteral administration, as well as to promote retention of thecompound at the site of injection. Non-limiting examples of oil-basedexcipients are known to those skilled in the art, includingpharmaceutical-grade sesame oil, soybean oil, castor oil, corn oil,cottonseed oil, peanut oil, Miglyol®, squalene oil, and the like. Theseoils may be purified, or refined, by a chromatography process to reducethe levels of polar impurities, thereby producing a USP-NF/JP/Ph.Eur.—grade products that possess consistent properties and impurityprofiles.

In some embodiments, the TLR7/8 agonist compound is initiallysolubilized in a 100% ethanol excipient and then diluted into the oil toa final concentration of between 2-20% ethanol to facilitatesolubilization of the compound in the oil. Ethanol suitable for use isone which does not contain water or denaturants, see e.g., 200 proofethanol, dehydrated alcohol USP-grade, etc.

In some embodiments, the pharmaceutical compositions comprise apreservative. Suitable preservatives include, for instance,antimicrobial agents and antioxidants. In preferred embodiments, thepharmaceutical composition is prepared under sterile conditions and isin a single use container, and thus does not necessitate inclusion of ananti-microbial agent. One skilled in the art will recognize thatpharmaceutical grade anti-oxidants, used to prevent coloring, odors, orperoxide formation, may be added to these oil-based formulations,including but not limited to butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), tertiary butylhydroquinone (TBHQ), vitamin E,propyl gallate, and the like. In certain embodiments, the addedantioxidant concentration in the formulation is at least 10 ppm, 50 ppm,100 ppm, 300 ppm, 500 ppm, and up to 1000 ppm, in order to ensurestability of the oil-based formulation for periods of time up to 1 yearwhen stored at temperatures from 5° C. to 40° C.

In some embodiments, the pharmaceutical compositions comprise anoil-in-water based nanoemulsion (see e.g., Dowling et al 2017 JCIInsight 2:e91020) or a liposome-based formulation (see e.g., VanHoevenet al 2017 Sci Rep 7:46426). In one embodiment exemplifying anoil-in-water nanoemulsion-based pharmaceutical composition, the TLR7/8agonist compounds of formula (J) or (K) are dissolved in an oil phasecomposed of phospholipids (e.g.,1,2-dimyristoyl-sn-glycero-3-phosphocholine;1,2-dipalmitoyl-sn-glycero-3-phosphocholine;1,2-distearoyl-sn-glycero-3-phosphocholine;1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol);1,2-dioleoyl-sn-glycero-3-phosphocholine; L-α-phosphatidylcholine; andthe like), triglyceride-based oils (see e.g., sesame oil, soybean oil,castor oil, corn oil, cottonseed oil, peanut oil, Miglyol®, squaleneoil, and the like), and optionally an organic modifier (e.g., ethanol).Next, an aqueous phase containing a suitable buffer, isotonic agent,emulsifying agent, and optionally a preservative is added to the oilphase and a crude emulsion is formed by high shear mixing (e.g.,Polytron®) for 5-10 minutes. Finally, a nanoemulsion containingparticles with a mean diameter in the range of 100-150 nm (assessed bydynamic light scattering), and dispersity index in the range of 0.1-0.2,is formed by processing the crude emulsion through a high shearhomogenizer (see e.g., Microfluidizer M110P) for 10-15 passes at 30,000psi.

In another embodiment exemplifying a liposome-based pharmaceuticalcomposition, the TLR7/8 agonist compounds of formula (J) or (K) aredissolved in organic modifiers and a suitable mixture of phospholipidsusing various ratios of neutrally-charged, positively-charged,negatively-charged, and PEGylated phospholipids, with varying lipid taillengths, and cholesterol (depending on the desired physiochemicalproperties of the liposome). The organic solvents are then removed usinga rotary evaporator and the lipid/compound film re-dissolved in anaqueous buffer until the formulation is translucent with no visibleparticles. Finally, the resulting multi-lamellar liposomes are processedusing either high shear homogenization or a membrane extruder (e.g.,Lipex®) into unilamellar liposomes with a mean diameter in the range of100-150 nm (assessed by dynamic light scattering) and dispersity indexin the range of 0.1-0.2. These examples are non-limiting, and oneskilled in the art will recognize that oil-in-water nanoemulsions andliposome-based pharmaceutical compositions can be formed by any of anumber of differing methods (see e.g., Brito et al 2013 Seminar Immunol25:130-145).

In some embodiments, the pharmaceutical compositions comprise a bulkingagent. Bulking agents are particularly useful when the pharmaceuticalcomposition is to be lyophilized before administration. In someembodiments, the bulking agent is a lyoprotectant that aids in thestabilization and prevention of degradation of the active agents duringfreeze-drying and/or during storage. Suitable bulking agents are sugars(mono-, di-, and polysaccharides) such as sucrose, lactose, trehalose,mannitol, sorbital, glucose, and raffinose.

In some embodiments, the pharmaceutical compositions comprise abuffering agent. Buffering agents control pH to inhibit degradation ofthe active agent during processing, storage, and optionallyreconstitution. Suitable buffers include, for instance, salts comprisingacetate, citrate, phosphate or sulfate. Other suitable buffers include,for instance, amino acids such as arginine, glycine, histidine, andlysine. The buffering agent may further comprise hydrochloric acid orsodium hydroxide. In some embodiments, the buffering agent maintains thepH of the composition within a range of 4 to 9. In some embodiments, thepH is greater than (lower limit) 4, 5, 6, 7, or 8. In some embodiments,the pH is less than (upper limit) 9, 8, 7, 6, or 5. That is, the pH isin the range of from about 4.0 to 9.0, in which the lower limit is lessthan the upper limit.

In some embodiments, the pharmaceutical compositions comprise a tonicityadjusting agent. Suitable tonicity adjusting agents include, forinstance, dextrose, glycerol, sodium chloride, glycerin, and mannitol.

Antigens

In one aspect, the present disclosure provides pharmaceuticalcompositions comprising an antigen. In some embodiments, thepharmaceutical composition comprises an alkyl chain modified1H-imidazo[4,5-c]quinoline TLR7/8 agonist, one or more excipients, andan antigen. In some of these embodiments, the antigen is a proteinantigen. In some of these embodiments, the antigen is a polysaccharideantigen, which is preferably covalently attached to a carrier protein.In some embodiments, the antigen is a microbial antigen, an allergen, ora tumor associated antigen. In some embodiments, the antigen is a viralantigen, a protozoan antigen, a bacterial antigen, or a fungal antigen.In some embodiments, the tumor antigen is a self-antigen or neoantigen.

In some embodiments, the pharmaceutical compositions comprise amicrobial antigen selected from the group consisting of a viral antigen,a bacterial antigen, a fungal antigen, and a parasite antigen. In someembodiments, the microbial antigen is from a microbe that causes aninfectious disease in a nonhuman, mammalian subject. In someembodiments, the microbial antigen is from a microbe that causes aninfectious disease in a human subject. In some embodiments, theinfectious disease is caused by a virus, a bacterium, a fungus, or aprotozoan parasite. Suitable microbial antigens include, for instance,antigens of adenovirus type 4, adenovirus type 7, Bacillus anthracis(anthrax), Mycobacterium tuberculosis, Corynebacterium diphtheriae(e.g., diphtheria toxoid), Clostridium tetani (e.g., tetanus toxoid),Bordetella pertussis, Haemophilus influenzae type B, hepatitis A virus,hepatitis B virus (e.g., HBsAg), human papillomavirus (types 6, 11, 16,18, 31, 33, 45, 52, and 58) influenza virus type A and B (e.g.,haemagglutinin, neuraminadase), influenza virus type B, parainfluenzavirus, Japanese encephalitis virus, measles virus, mumps virus, rubellavirus, Neisseria menigitidis (Groups A, B, C, Y, and W-135),Streptococcus pneumoniae (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C,19A, 19F, and 23F), poliovirus, rabies virus, rotavirus, vaccinia virus,Salmonella typhi, varicella zoster virus, and yellow fever virus (see,e.g., Plotkin, S. A., Orenstein, W., Offit, P. A., Edwards K. M. (2017).Plotkin's Vaccines, 7^(th) edition. Elsevier). In some embodiments, themicrobial antigen is a viral antigen of Herpes simplex virus type 1 or2, human herpes virus, human immunodeficiency virus type 1, andrespiratory syncytial virus. In some embodiments, the microbial antigenis a fungal antigen of Candida albicans, Aspergillus flavus,Cryptococcus neoformans, Histoplasma capsulatum, and Pneumocystiscarinii. In some embodiments, the microbial antigen is a parasiteantigen of a Leishmania species, a Plasmodium species, a Schistosomaspecies, or a Trypanosoma species.

In some embodiments, the pharmaceutical compositions comprise anallergen. In some embodiments, the allergen is an environmental antigensuch as mammalian, insect, plant, and mold allergens. In someembodiments, the mammalian allergen includes fur and dander. Suitablemammalian allergens include, for instance, cat Fel d 1, cow Bos d 2, dogCan f I, and Can f II, horse Equ c 1, and mouse MUP. In someembodiments, the insect allergen includes insect feces and venom.Exemplary insect allergens include ant Sol i2, bee PLA and Hya,cockroach Bla g Bd9OK, Bla g4, GST, and Per a3, dust mite Der p2, Derf2, Der p10, and Tyr p2, hornet Dol m V, mosquito Aed a 1, and yellowjacket hyaluronidase and phospholipase. In some embodiments, the plantallergen includes grass, weed, and tree allergens (e.g., pollens).Suitable grass allergens include, for instance, allergens of Kentuckybluegrass, meadow fescue, orchard grass, redtop grass, perennialryegrass, sweet vernal grass, and timothy. Exemplary plant allergensinclude barley Hor v 9, birch Bet v1 and v2, cherry Pru a 1, corn Zml3,grass Phl p 1, 2, 4, 5, 6, 7, 11, and 12, Hol 1 5, Cyn d 7 and d12,cedar Jun a 2, Cry j 1, and j2, juniper Jun o2, latex Hey b7, yellowmustard Sin a I, rapeseed Bra r 1, ragweed Amb a 1, and rye Lol p1. Insome embodiments, the mold allergen is an Aspergillus fumigatus allergensuch as Asp f 1, 2, 3, 4, and 6. In some embodiments, the allergen is afood allergen such as a shell fish allergen, a legume allergen, a nutallergen or a milk allergen. Exemplary food allergens include shrimptropomyosin, peanut Ara h 1, 2, 3, 8, and 9, walnut Jug r 1 and 3,hazelnut Cor a 1, 14, and 8 LTP, cow's milk lactalbumin, casein, andlactoferrin.

In some embodiments, the pharmaceutical compositions comprise a tumorantigen. In some embodiments, the tumor antigen comprises the amino acidsequence of a full length protein or a fragment thereof (e.g., apolypeptide of about 10 to about 100 amino acids in length). In someembodiments, the tumor antigen comprises a full length protein orpolypeptide fragment of one or more of the group consisting of WT1,MUC1, LMP2, HPV E6, HPV E7, EGFRvIII, Her-2/neu, idiotype, MAGE A3, p53,NY-ESO-1 (CTAG1), PSMA, CEA, MelanA/Mart1, Ras, gp100, proteinase 3,bcr-able, tyrosinase, survivin, PSA, hTERT, sarcoma translocationbreakpoints, EphA2, PAP, MP-IAP, AFP, EpCAM, ERG, NA17-A, PAX3, ALK,androgen receptor, cyclin B1, MYCN, PhoC, TRP-2, mesothelin, PSCA, MAGEA1, CYP1B1, PLAC1, BORIS, ETV6-AML, NY-BR-1, RGSS, SART3, carbonicanhydrase IX, PAXS, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4,SSX2, XAGE 1, B7-H3, legumain, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PAP,PDGFR-beta, MAD-CT-2, CEA, TRP-1 (gp75), BAGE1, BAGE2, BAGE3, BAGE4,BAGE5, CAMEL, MAGE-A2, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A8, MAGE-A9,MAGE-A10, MAGE-A11, MAGE-A12, and Fos-related antigen 1. In somepreferred embodiments, the tumor antigen comprises an amino acidsequence or fragment thereof from one or more of the group consisting ofgp100, hTERT, MAGE A1, MAGE A3, MAGE A10, MelanA/Mart1, NY-ESO-1, PSA,Ras, survivin, TRP1 (gp75), TRP2, and tyrosinase.

IV. METHODS OF USE

The pharmaceutical compositions of the present disclosure are suitablefor a plurality of uses involving stimulating an immune response in amammalian subject in need thereof. Mammalian subjects include, but arenot limited to, humans, nonhuman primates, rodents, pets, and farmanimals. In some embodiments, the pharmaceutical compositions areadministered to the subject in an amount effective to achieve a specificoutcome.

Dosage and Mode of Administration

As with all pharmaceutical compositions, the effective amount and modeof administration may vary based on several factors evident to oneskilled in the art. Factors to be considered include potency of thealkyl chain modified 1H-imidazo[4,5-c]quinoline TLR7/8 agonist compound,ability of the compound and pharmaceutical composition to promoteretention of the agonist compound at the site of administration, theroute of administration, and whether the pharmaceutical compositioncontains an antigen. Other factors to be considered include the diseasemodification outcome to be achieved, and the number/frequency of dosesto be administered during a therapeutic regimen.

A suitable dosage range is one that provides the desired clinicaleffect. Dosage may be determined by the amount of the TLR7/8 agonist inthe pharmaceutical composition that needs to be administered to asubject to yield a desired therapeutic response with minimal adverseevents. An exemplary dosage range of the TLR7/8 agonist compound givenin an amount to be delivered by subject weight is from about 1 to 5000ng/kg, such as about 1 to 2,500 ng/kg, about 1 to 1,000 ng/kg, about 1to 500 ng/kg, about 1 to 250 ng/kg, about 1 to 100 ng/kg, about 1 to 50ng/kg, about 50 to 2,500 ng/kg, about 50 to 1,000 ng/kg, about 50 to 500ng/kg, about 100 to 5,000 ng/kg, about 100 to 2,500 ng/kg, about 100 to1,000 ng/kg, about 100 to 500 ng/kg, about 500 to 5,000 ng/kg, about1,000 to 5,000 ng/kg, about 2,000 to 5,000 ng/kg, about 2,500 to 5,000ng/kg, about 3,000 to 5,000 ng/kg, or about 4,000 to 5,000 ng/kg. Insome embodiments, the dosage is greater than about (lower limit) 1, 5,10, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 ng/kg. In someembodiments, the dosage is less than about (upper limit) 5000, 2000,1000, 900, 800, 700, 600, 500, 450, 400, 350, 300, 250, 200, 150, or 100ng/kg. That is, the dosage is anywhere in the range of from about 1 to5000 ng/kg in which the lower limit is less than the upper limit. Anexemplary dosage range of the TLR7/8 agonist given in an amount to bedelivered to a subject is from about 1 to 5000 ng. In some embodiments,the dosage can be even higher, for example, about 2,500 to 500,000ng/kg, about 5,000 to 500,000 ng/kg, about 2,500 to 150,000 ng/kg, about2,500 to 100,000 ng/kg, about 2,500 to 50,000 ng/kg, about 2,500 to25,000 ng/kg, about 2,500 to 10,000 ng/kg, about 10,000 to 500,000ng/kg, about 25,000 to 500,000 ng/kg, about 50,000 to 500,000 ng/kg,about 100,000 to 500,000 ng/kg, or about 150,000 to 500,000 ng/kg.

In some embodiments, when the pharmaceutical composition furthercomprises an antigen, the antigen dosage range given in an amount to bedelivered to a subject is from about 1 μg to 500 μg. In someembodiments, the antigen dosage is from about 1 μg to 50 μg. In someembodiments, the antigen dosage is greater than about (lower limit) 1,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 400 μg. In someembodiments, the antigen dosage is less than about (upper limit) 500,400, 300, 200, 100, 50, 45, 40, 35, 30, 25, 20, 15, or 10 μg. That is,the antigen dosage is anywhere in the range of from about 1 to 500 μg inwhich the lower limit is less than the upper limit. The optimum antigendosage can be determined by experimental means for each individualantigen.

Likewise, a suitable route of administration is one that provides thedesired effect. In general, the pharmaceutical compositions of thepresent disclosure are intended for parenteral administration (e.g., notoral or rectal administration). Suitable routes of administrationinclude injection, topical, and inhalation. In particular, thepharmaceutical compositions of the present disclosure may beadministered by a route such as intratumoral, intramuscular,subcutaneous, intravenous, epidermal (gene gun), transdermal, andinhalation. Devices suitable for administration by inhalation include,for instance, atomizers, vaporizers, nebulizers, and dry powderinhalation delivery devices. In some embodiments, when thepharmaceutical compositions are intended to treat a solid tumor, thecompositions are administered intratumorally. In one embodiment,intratumoral administration is by injection into at least one tumorlesion.

A suitable dosing regimen of the TLR7/8 agonist formulated in thepharmaceutical composition is one that provides the desired effect in aprophylactic or therapeutic context with minimal adverse events. Thenumber of doses administered by a chosen route may be one or more thanone. Frequency of dosing may range from weekly, bi-weekly, monthly,bi-monthly, or 3 to 12 months between doses. An exemplary dose frequencyof the TLR7/8 agonist is from about once per week to once every 8 weeks.In some embodiments, the dose frequency is greater than about (upperlimit) once every 8, 6, 4, 2, or 1 weeks. In some embodiments, the dosefrequency is less than about (lower limit) once every 7, 10, or 14 days.An exemplary dose frequency range of the TLR7/8 agonist to be deliveredto a subject is from about once every week to once every 4 weeks. Insome embodiments, 2 doses are administered, with the second dose beingadministered one to two months after the first dose. In someembodiments, 3 doses are administered, with the second dose beingadministered one to two months after the first dose and the third dosebeing administered one to five months after the second dose. In otherembodiments, a series of doses may be administered over a 3 to 12 monthtreatment schedule, where the dose frequency is once every week, everyother week, every third week, or monthly. In other embodiments, ashorter or longer period of time may elapse between doses. In certainembodiments, the interval between successive dosages may vary in termsof number of weeks or number of months. In one embodiment, a series of2, 3, 4, 5, or 6 weekly doses may be administered followed by a secondseries of weekly doses at a later time point. One skilled in the artwill be able to adjust the dosage regimen by measuring biologicaloutcomes such as antigen-specific antibody responses or tumorregression.

Stimulation of an Immune Response

In one aspect, the present disclosure provides methods of stimulating animmune response in a mammalian subject in need thereof, comprisingadministration to a mammalian subject a pharmaceutical composition in anamount and frequency sufficient to stimulate an immune response in saidsubject. “Stimulating” an immune response means increasing the immuneresponse, which can arise from eliciting a de novo immune response(e.g., as a consequence of an initial vaccination regimen) or enhancingan existing immune response (e.g., as a consequence of a boostervaccination regimen). In some embodiments, stimulating an immuneresponse comprises one or more of the group consisting of: stimulatingIFNα production, stimulating production of Type 1 and/or Type 2interferons, stimulating IL-6 production, stimulating TNFα production,stimulating B lymphocyte proliferation, stimulating interferonpathway-associated gene expression, stimulatingchemoattractant-associated gene expression, and stimulating plasmacytoiddendritic cell (pDC) or myeloid dendritic cell (mDC) maturation. Methodsfor measuring stimulation of an immune response are known in the art anddescribed in the biological examples of the present disclosure. Inembodiments in which the pharmaceutical composition further comprises anantigen, stimulating an immune response comprises inducing anantigen-specific antibody response.

For instance, in some embodiments in which the pharmaceuticalcomposition further comprises an antigen, the present disclosureprovides methods of inducing an antigen-specific antibody and/or T cellresponse in a mammalian subject in need thereof by administering thepharmaceutical composition in an amount sufficient to induce anantigen-specific antibody and/or T cell response in said subject.“Inducing” an antigen-specific antibody response means increasing titersof antigen-specific antibodies above a threshold level, such as apre-administration baseline titer or a seroprotective level. “Inducing”an antigen-specific T cell response means stimulating antigen-specificcytotoxic T lymphocytes, generating antigen-specific T cells that hometo non-immunized tumor sites, generating T cells that are less exhaustedand/or enhancing the immune response to additional tumor antigens byepitope spreading.

Analysis (both qualitative and quantitative) of the immune response canbe by any method known in the art, including, but not limited to,measuring antigen-specific antibody production (including measuringspecific antibody subclasses), activation of specific populations oflymphocytes such as B cells and helper T cells, measuring expression ofa set of genes specific to a particular immune cell type, production ofcytokines such as IFNα, IL-6, IL-12, IL-18, TNFα, and/or release ofhistamine from basophils or mast cells. Methods for measuringantigen-specific antibody responses include enzyme-linked immunosorbentassay (ELISA). Production of cytokines can also be measured by ELISA.Gene expression analysis can be performed by TaqMan® or nCounter® geneexpression assays. Activation of specific populations of lymphocytes canbe measured by proliferation assays and with fluorescence-activated cellsorting (FACS).

Preferably, a Th1-type immune response is stimulated (i.e., elicited orenhanced). With reference to the present disclosure, stimulating aTh1-type immune response can be determined in vitro or ex vivo bymeasuring cytokine production from cells treated with an active agent ofthe present disclosure (alkyl chain derivatives of1H-imidazo[4,5-c]quinolines that are TLR7/8 agonists) as compared tocontrol cells not treated with the active agent. Examples of “Th1-typecytokines” include, but are not limited to, IL-2, IL-12, IFNγ, and IFNα.In contrast, “Th2-type cytokines” include, but are not limited to, IL-4,IL-5, and IL-13. Cells useful for the determination of immunostimulatoryactivity include cells of the immune system such as antigen presentingcells, lymphocytes, and preferably macrophages and T cells. Suitableimmune cells include primary cells such as peripheral blood mononuclearcells, including pDCs, monocytes, mDCs, and B cells, or splenocytesisolated from a mammalian subject.

Stimulating a Th1-type immune response can also be determined in amammalian subject treated with an active agent of the present disclosureby measuring levels of IL-2, IL-12, and interferon either before andafter administration, or as compared to a control subject not treatedwith the active agent. Stimulating a Th1-type immune response can alsobe determined by measuring the ratio of Th1-type to Th2-type antibodytiters. “Th1-type” antibodies include human IgG1 and IgG3, and murineIgG2a. In contrast, “Th2-type” antibodies include human IgG2, IgG4, andIgE, and murine IgG1 and IgE.

Treatment of Disease

The present disclosure further provides methods of preventing aninfectious disease in a mammalian subject in need thereof, comprisingadministration of a pharmaceutical composition in an amount sufficientto prevent an infectious disease in said subject. That is, in someembodiments, the present disclosure provides prophylactic vaccines. Insome embodiments, the mammalian subject is at risk of exposure to aninfectious agent. “Preventing” an infectious disease means to protect asubject from developing an infectious disease. In some embodiments,preventing an infectious disease further comprises protecting a subjectfrom being infected with an infectious agent (e.g., protecting a subjectfrom developing an acute or a chronic infection). Additionally, thepresent disclosure provides methods of ameliorating a symptom of aninfectious disease in a mammalian subject in need thereof, comprisingadministration of a pharmaceutical composition in an amount sufficientto ameliorate a symptom of an infectious disease in said subject. Thatis, in some embodiments the present disclosure provides therapeuticvaccines. In some embodiments, the subject is acutely or chronicallyinfected with an infectious agent. The infectious disease may be a viral(e.g., hepatitis, herpes or human papilloma viruses), bacterial, fungal,or parasitic disease. In some embodiments, the pharmaceuticalcomposition further comprises a viral, bacterial, fungal, or parasiticantigen. “Ameliorating” a symptom of an infectious disease means toimprove a symptom, preferably diminishing the extent of the disease.

Moreover, the present disclosure provides methods of ameliorating asymptom of an IgE-related disorder in a mammalian subject in needthereof, comprising administration of a pharmaceutical composition in anamount sufficient to ameliorate a symptom of an IgE-related disorder insaid subject. In some preferred embodiments, the IgE-related disorder isan allergy. Allergies include, but are not limited to, allergic rhinitis(hay fever), sinusitis, eczema, and hives. In some embodiments, thepharmaceutical composition further comprises an allergen. “Ameliorating”a symptom of an IgE-related disorder means to improve a symptom,preferably diminishing the extent of the disorder. For instance, if theIgE-related disorder is allergic rhinitis, ameliorating a symptom meansto reduce swelling of nasal mucosa, reduce rhinorrhea (runny nose),and/or reduce sneezing.

Furthermore, the present disclosure provides a plurality of methods oftreating cancer in a mammalian subject in need thereof, comprisingadministration of a pharmaceutical composition in an amount sufficientto treat cancer in said subject. In certain embodiments, the presentdisclosure provides methods of treating cancer in a mammalian subject inneed thereof, comprising administering an effective amount of apharmaceutical composition by intratumoral delivery. In another aspectof the method, intratumoral delivery comprises injection of thepharmaceutical composition into at least one tumor lesion. In otheraspects, treating cancer comprises inducing accumulation of tumorantigen-specific T cells in the injected tumor, for example, at greaternumbers than had the pharmaceutical composition been administered at anextratumoral site. In other aspects, treating cancer comprises elicitinga systemic, tumor antigen-specific T cell response, including forexample, a systemic, tumor antigen-specific T cell response of a highermagnitude than had the immunogenic composition been administered at anextratumoral site. In other aspects, treating cancer comprises elicitinga systemic tumor antigen-specific T cell response. In other aspects,treating cancer comprises reducing numbers of CD4+ FoxP3+ regulatory Tcells in the injected tumor. In other aspects, the subject has one ormore uninjected tumors (primary or metastatic lesions) in addition tothe injected tumor, and treating cancer comprises one or more of thefollowing: (a) reducing the number of uninjected tumors; (b) reducingthe volume of uninjected tumors; and (c) retarding the growth ofuninjected tumors. In some aspects, treating cancer comprises one ormore of the following: (d) increasing the survival time of the subject;(e) reducing the volume of the injected tumor; and (f) retarding thegrowth of the injected tumor. In some embodiments, when the cancer is asolid tumor, “treating” cancer comprises shrinking the size of the solidtumor and any metastatic lesions, or otherwise reducing viable cancercell numbers. In other embodiments, when the cancer is a solid tumor,“treating” cancer comprises delaying growth of the solid tumor and anymetastatic lesions. In some aspects, treating cancer comprisesincreasing progression free survival or increasing time to progression.In other embodiments, the method further comprises administering aneffective amount of a second, or additional, therapeutic agents to thesubject. “Treating” cancer means to bring about a beneficial clinicalresult, such as causing remission or otherwise prolonging survival ascompared to expected survival in the absence of treatment. In somepreferred embodiments, “treating cancer” comprises assessing a patient'sresponse to the immunogenic composition according to the ResponseEvaluation Criteria in Solid Tumors (RECIST version 1.1) as described(see, e.g., Eisenhauer et al 2009 Eur J Cancer 45:228-247). Responsecriteria to determine objective anti-tumor responses per RECIST include:complete response, partial response, progressive disease, and stabledisease.

In some embodiments, the tumor is a sarcoma, a carcinoma, or an actinickeratosis. In some embodiments, the tumor is a lymphoma. In someembodiments, the cancer is selected from the group consisting of breastcancer, prostate cancer, lung cancer, colorectal cancer, uterine cancer,bladder cancer, melanoma, head and neck cancer, non-Hodgkin lymphoma,kidney cancer, ovarian cancer, pancreatic cancer, and thyroid cancer. Insome embodiments, the cancer is a primary cancer of a site selected fromthe group consisting of oral cavity, digestive system, respiratorysystem, skin, breast, genital system, urinary system, ocular system,nervous system, endocrine system, and lymphoma.

In some embodiments, the method further comprises administering aneffective amount of a second therapeutic agent to the subject. In someof these embodiments, the second therapeutic agent comprises achemotherapeutic agent selected from the group consisting ofactinomycin, afatinib, alectinib, asparaginase, azacitidine,azathioprine, bicalutamide, binimetinib, bleomycin, bortezomib,camptothecin, carboplatin, capecitabine, carmustine, certinib,cisplatin, chlorambucil, cobimetinib, crizotinib, cyclophosphamide,cytarabine, dabrafenib, dacarbazine, daunorubicin, docetaxel,doxifluridine, doxorubicin, encorafenib, erlotinib, epirubicin,epothilone, etoposide, fludarabine, flutamine, fluorouracil, gefitinib,gemcitabine, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan,lapatinib, letrozole, mechlorethamine, mercaptopurine, methotrexate,mitomycin, mitoxantrone, octreotide, oxaliplatin, paclitaxel,pemetrexed, raltitrexed, sorafenib, sunitinib, tamoxifen, temozolomide,teniposide, tioguanine, topotecan, trametinib, valrubicin, vemurafenib,vinblastine, vincristine, vindesine, vinorelbine, and combinationsthereof. In some embodiments, the second therapeutic agent comprises oneor both of a BRAF inhibitor and a MEK inhibitor. In some embodiments,the second therapeutic agent comprises a epigenetic modulator selectedfrom the group consisting of HDAC inhibitors (see e.g., voronistat[SAHA], romidepsin, entinostat, abexinostat, elinostat [CHR-3996],panobinostat, quisinostat [JNJ-26481585],4SC-202, resminostat [SB939],pracinostat [CI-9940], and valproate), DNA methyltransferase inhibitors(see e.g., azacytidine, decitabine, zebularine, SGI-1027, RG-108, andsinfungin), and combinations thereof.

In some of these embodiments, the second therapeutic agent is anantagonist of an inhibitory immune checkpoint molecule, for example, aninhibitory immune checkpoint molecule selected from the group consistingof PD-1, PD-L1, PD-L2, CTLA-4 (CD152), LAG-3, TIM-3, TIGIT, IL-10,indoleamine 2,3-dioxygenase (IDO), P-selectin glycoprotein ligand-1(PSGL-1), and TGF-beta. In some of these embodiments, the secondtherapeutic agent is an agonist of an immune stimulatory molecule. Insome of these embodiments, the immune stimulatory molecule is selectedfrom the group consisting of CD27, CD40, OX40 (CD134), GITR, 4-1BB(CD137), CD28, and ICOS (CD278). In some of these embodiments, thesecond therapeutic agent comprises an antibody, fragment, or derivativethereof. In some of these embodiments, the second therapeutic agent isan antagonist of an inhibitory immune checkpoint molecule and the secondtherapeutic agent comprises an antibody, fragment, or derivativethereof. In some embodiments, the method further comprises administeringradiation therapy and/or administering an effective amount of a secondtherapeutic agent to the subject. In some of these embodiments, theeffective amount of the immunogenic composition and the effective amountof the second therapeutic agent together result in an additive effect orbetter against the tumor. In some of these embodiments, the effectiveamount of the immunogenic composition and the effective amount of thesecond therapeutic agent together result in a synergistic effect againstthe tumor.

In some embodiments of the method, treating infectious disease or cancerdoes not result in development of flu-like symptoms of such severitythat repeated administration of the immunogenic composition iscontraindicated, wherein the flu-like symptoms comprise one or more ofthe group consisting of fever, headache, chills, myalgia, and fatigue.

In some embodiments, the present disclosure provides kits that comprisea pharmaceutical composition (e.g., a TLR7/8 agonist compound of formula(K), an excipient or excipients, and optionally an antigen) and a set ofinstructions relating to the use of the composition for the methodsdescribed herein. The pharmaceutical composition of the kits is packagedappropriately. If the pharmaceutical composition is a liquid or asuspension of nanoparticles, a silicon dioxide vial (e.g., SCHOTT Type Iplus®) with a rubber stopper (e.g., Exxpro halobutyl elastomer) and analuminum crimp-top is typically used as the container-closure system. Insome embodiments, the kits further comprise a device (e.g., syringe andneedle) for administration of the pharmaceutical composition. In otherembodiments, the kits further comprise a pre-filled syringe/needlesystem, autoinjectors, or needleless devices. The instructions relatingto the use of the pharmaceutical composition generally includeinformation as to dosage, schedule, and route of administration for theintended methods of use.

V. EXAMPLES

Although the present disclosure has been described in some detail by wayof illustration and example for purposes of clarity and understanding,it will be apparent to those skilled in the art that certain changes andmodifications may be practiced. Therefore, the following synthetic andbiological examples should not be construed as limiting the scope of thepresent disclosure, which is delineated by the appended claims.

Synthetic Examples Example S1: Synthesis ofN-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)tetradecanamide(Compound No. 63-10)

Part A. Nitric acid (125 mL) was added to a slurry of quinoline-2,4-diol(50 g, 0.3 mole) in acetic acid (500 mL), and the reaction mixture washeated to 65° C. for three hours. The mixture was then cooled to 5-10°C. and the solid material collected by filtration, washed with coldwater, and air dried. The resulting solid was then recrystallized frommethanol and dried under vacuum to yield 58 g of3-nitro-2,4-quinolinediol as a yellow solid.

Part B. Phosphorus oxychloride (150 mL) was added to3-nitro-2,4-quinolinediol (58 g) under an argon atmosphere and heated to95° C. for 4 hours. The mixture was then cooled to room temperature andpoured onto crushed ice with constant stirring. The precipitated productwas collected by filtration, washed with water, and dried under vacuum.The crude solid was purified by flash chromatography over silica gelusing hexane/ethyl acetate to yield 35 g of2,4-dichloro-3-nitroquinoline.

Part C. Tert-butyl (4-(aminomethyl)benzyl)carbamate (37.3 g, 0.16 mole,1.1 eq) was added to a solution of 2,4-dichloro-3-nitroquinoline (35 g,0.15 mole, 1.0 eq) in anhydrous dichloromethane (400 mL) andtrimethylamine (16.0 g, 0.16 mole, 1.1 eq), and stirred overnight atroom temperature. The solvents were removed under reduced pressure andthe crude product was purified by flash chromatography over silica gelusing hexane/ethyl acetate to yield 52 g of tert-butyl(4-(((2-chloro-3-nitroquinolin-4-yl)amino)methyl)benzyl)carbamate.

Part D. A solution of tert-butyl(4-(((2-chloro-3-nitroquinolin-4-yl)amino)methyl)benzyl)carbamate (52 g,0.12 mole) in ethyl acetate (250 mL) was hydrogenated in the presence of5% platinum on carbon (2.0 g) and sodium sulfate (52 g) using a Parrhydrogenation apparatus at 60 psi for 12 hours. The platinum catalystand sodium sulfate were removed by filtering through a pad of Celite®,and the filtrate concentrated under reduced pressure. The product wasfurther purified by flash chromatography over silica gel eluting withhexane/ethyl acetate to yield 32 g of tert-butyl(4-(((3-amino-2-chloroquinolin-4-yl)amino)methyl)benzyl)carbamate.

Part E. Pentanoyl chloride (9.7 mL, 81.5 mmol, 1.05 eq) was added slowlyto a solution of tert-butyl(4-(((3-amino-2-chloroquinolin-4-yl)amino)methyl)benzyl)carbamate (32 g,77.6 mmol, 1.0 eq) in anhydrous tetrahydrofuran (350 mL) and pyridine(30 mL) at 0-5° C. The reaction mixture was then warmed to roomtemperature and stirred for 12 hours. The solvents were removed underreduced pressure and then the solids were re-dissolved in ethyl acetate(400 mL), washed successively with water and saturated sodiumbicarbonate (150 mL), and finally dried over anhydrous magnesiumsulfate. The product was further purified by flash chromatography oversilica gel, eluting with hexane/ethyl acetate to yield 22 g oftert-butyl(4-(((3-butyramido-2-chloroquinolin-4-yl)amino)methyl)benzyl)carbamate.

Part F. Water (80 mL) was added to a solution of tert-butyl(4-(((3-butyramido-2-chloroquinolin-4-yl)amino)methyl)benzyl)carbamate(22 g, 44.2 mmol, 1.0 eq) in ethanol (320 mL), followed by the additionof potassium carbonate (12.2 g, 88.4 mmol, 2.0 eq), and the mixture washeated with vigorous stirring to 55° C. for 16 hours. This reactionmixture was concentrated and the residue was partitioned between ethylacetate (500 mL) and water (250 mL). The ethyl acetate layer was thenwashed with water (100 mL), dried over anhydrous magnesium sulfate, andconcentrated under reduced pressure. The product was further purified byflash chromatography over silica gel, eluting with hexane/ethyl acetateto yield 15.4 g of tert-butyl(4-((2-butyl-4-chloro-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)carbamate.

Part G. Tert-butyl(4-((2-butyl-4-chloro-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)carbamate(15.4 g, 32.2 mmol, 1 eq) was dissolved in anhydrous dimethyl formamide(125 mL), and then sodium azide (8.4 g, 128.6 mmol, 4 eq) was added tothis solution. The resulting suspension was degassed and stirred underargon atmosphere at 110-115° C., with the reaction progress beingmonitored by reverse-phase HPLC analysis. After 18 hours, the reactionmixture was cooled to room temperature, poured into cold water (500 mL),and extracted with ethyl acetate (3×100 mL). The combined extract waswashed with water (2×75 mL), dried over magnesium sulfate, filteredthrough Celite, and concentrated under reduced pressure to yield anoff-white solid. This solid was further worked up by re-crystallizationwith 1:1 ethyl/hexane to yield 12.5 g of tert-butyl(4-((4-azido-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)carbamate.

Part H. Tert-butyl(4-((4-azido-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)carbamate(12.5 g, 25.7 mmol) was added to concentrated hydrochloric acid (65 mL),and 10% platinum on carbon (3.0 g) was added to this suspension. Thisreaction mixture was subjected to hydrogenation at 65 psi, with thereaction progress being monitored by reverse-phase HPLC analysis. After6 days, the catalyst was filtered off and the filtered cake was washedwith water (2×25 mL). The cake was cooled in an ice bath, ice cold 1Nsodium hydroxide was added drop wise while stirring vigorously until thepH reached 8.5, and the material was extracted with dichloromethanecontaining 5% methanol (4×75 mL). The combined extract was dried overmagnesium sulfate, filtered, and concentrated under reduced pressure.The residue was purified on a silica gel column using 8%methanol/dichloromethane containing 1% aqueous ammonia to yield 5.3 g of1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine.

Part I. Myristic acid (27.4 mg, 0.12 mmol, 1.2 eq) and trimethylamine(0.2 mL) were added to a solution of1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (34mg, 0.1 mmol, 1.0 eq) in anhydrous dimethylformamide (2 mL), and theslurry was mixed for 5 minutes followed by the addition of HBTU (47.4mg, 0.125 mmol, 1.25 eq). This reaction mixture was further stirred for2 hours under argon atmosphere. The solvent was removed under reducedpressure, the residue dissolved in ethyl acetate (30 mL) and washed withwater (2×10 mL), then dried using magnesium sulfate and concentratedunder vacuum. This product was purified using column chromatography (6%methanol/dichloromethane) to yield 35 mg ofN-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)tetradecanamide(Compound No. 63-10). Product purity was assessed to be ˜98% byreverse-phase HPLC, the intended synthetic mass of 569.8 was confirmedby LC/MS, and the intended synthetic structure confirmed by 300 MHzproton NMR (CDCl₃): δ 7.98 (d, J=8.1 Hz, 1H), 7.75 (d, J=8.4 Hz, 1H),7.67 (t, J=8.4 Hz, 1H), 7.40 (t, J=7.5 Hz, 1H), 7.30 (d, J=8.1 Hz, 2H),7.06 (d, J=8.1 Hz, 2H), 5.95 (s, 2H), 4.33 (s, 2H), 3.74 (s, 2H), 3.01(t, J=7.8 Hz, 2H), 2.20 (t, J=7.5 Hz, 2H), 1.82-1.9 (m, 2H), 1.42-1.70(m, 4H), 1.26-1.48 (m, 20 H), 0.85-1.05 (m, 6H).

Example S2: Synthesis of2-butyl-1-(4-((pentylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine(Compound No. 63-17)

Parts A-H were the same as in Example S1.

Part I. Valeric acid (34 mg, 0.33 mmol, 1.2 eq) and trimethylamine (140mg, 1.39 mmol, 5.0 eq) were added to a solution of1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (100mg, 0.28 mmol, 1.0 eq) in anhydrous dimethylformamide (2 mL), and theslurry was mixed for 5 minutes followed by the addition of HBTU (131 mg,0.34 mmol, 1.25 eq). This reaction mixture was further stirred for 2hours under an argon atmosphere. The reaction was diluted with ethylacetate (100 mL) and washed with water (3×30 mL), then dried usingmagnesium sulfate and concentrated under vacuum. The crude residue wastaken up in ethyl acetate and methanol and purified using columnchromatography (6% methanol/dichloromethane) to yield 160 mg ofN-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)pentanamide.

Part J. A solution of borane-dimethyl sulfide complex (2.0 M, 1.5 mL,excess) was added to solution ofN-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)pentanamide(127 mg, 0.28 mmol, 1.0 eq) in anhydrous tetrahydrofuran (5 mL) at roomtemperature, and the reaction mixture was heated to reflux for 12 hours.The mixture was cooled to ambient temperature, quenched with 3N HCl (1mL), and stirred for 4 hours. The pH of the reaction mixture was madealkaline by the addition 2N sodium hydroxide and the product wasextracted with dichloromethane (20 mL×10). The combined organic layerswere concentrated under reduced pressure and the residue purified byflash chromatography with 6% methanol/dichloromethane as an eluent toyield 24 mg of2-butyl-1-(4-((pentylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine(Compound No. 63-17). Product purity was assessed to be ˜96% byreverse-phase HPLC, the intended synthetic mass of 429.6 was confirmedby LC/MS, and the intended synthetic structure confirmed by 300 MHzproton NMR (CDCl₃): δ 7.8 (d, J=8.4 Hz, 1H), 7.70 (d, J=8.5 Hz, 1H),7.45 (t, J=8.4, 1H), 7.34 (d, J=8.5 Hz, 2H), 7.10 (t, J=8.4 Hz, 1H),7.02 (d, J=8.3 Hz, 2H), 5.70 (br s, 4H), 3.80 (s, 2H), 2.89 (t, J=7.6Hz, 2H), 2.62 (t, J=7.2 Hz, 2H), 1.75-1.84 (m, 2H), 1.2-1.75 (m, 8H),0.65-1.0 (m, 6H).

Example S3: Synthesis of2-butyl-1-(4-(((2-cyclopropylethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine(Compound No. 63-33)

Parts A-H were the same as in Example S1.

Part I. Cyclopropylacetic acid (33 mg, 0.33 mmol, 1.2 eq) andtrimethylamine (140 mg, 1.39 mmol, 5.0 eq) were added to a solution of1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (100mg, 0.28 mmol, 1.0 eq) in anhydrous dimethylformamide (2 mL), and theslurry was mixed for 5 minutes followed by the addition of HBTU (131 mg,0.34 mmol, 1.25 eq). This reaction mixture was further stirred for 2hours under an argon atmosphere. The reaction was diluted with ethylacetate (100 mL) and washed with water (3×30 mL), then dried usingmagnesium sulfate and concentrated under vacuum. The crude residue wastaken up in ethyl acetate and methanol, and purified using columnchromatography (6% methanol/dichloromethane) to yield 140 mg ofN-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-2-cyclopropylacetamide(Compound No. 63-34).

Part J. A solution of borane-dimethyl sulfide complex (2.0 M, 1.5 mL,excess) was added to solution ofN-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-2-cyclopropylacetamide(123 mg, 0.28 mmol, 1.0 eq) in anhydrous tetrahydrofuran (5 mL) at roomtemperature, and the reaction mixture was heated to reflux for 12 hours.The mixture was cooled to ambient temperature, quenched with 3N HCl (1mL), and stirred for 4 hours. The pH of the reaction mixture was madealkaline by the addition 2N sodium hydroxide and the product wasextracted with dichloromethane (20 mL×10). The combined organic layerswere concentrated under reduced pressure and the residue purified byflash chromatography with 6% methanol/dichloromethane as an eluent toyield 28 mg of2-butyl-1-(4-(((2-cyclopropylethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine(Compound No. 63-33). Product purity was assessed to be 97% byreverse-phase HPLC, the intended synthetic mass of 427.6 was confirmedby LC/MS, and the intended synthetic structure confirmed by 400 MHz ¹HNMR (CDCl₃): δ 7.80 (dd, J=8.5, 1.0 Hz, 1H), 7.70 (dd, J=8.3, 1.1 Hz,1H), 7.42 (m, J=8.4. 7.0, 1.4 Hz, 1H), 7.29 (as, 1H), 7.25 (as, 1H),7.10 (m, J=8.2, 7.1, 1.3 Hz, 1H), 7.00 (d, J=8.4 Hz, 2H), 5.92 (bs, 2H),5.69 (s, 2H), 3.75 (s, 2H), 2.86 (dd, J=8.0 Hz, 2H), 2.68 (dd, J=8.0 Hz,2H), 1.82-1.74 (m, 2H), 1.45-1.36 (m, 4H), 0.91 (t, J=7.8 Hz, 3H),0.91-0.85 (m, 1H), 0.68-0.60 (m, 1H), 0.42-0.37 (m, 2H), 0.04-0.00 (m,2H).

Example S4: Synthesis of Other Exemplary Compounds

Exemplary N-alkyl compounds of formula (J-1) were synthesized usingsimilar procedures as for Compound No. 63-33. Using techniques known tothose skilled in the art, the calculated partitioning coefficient(cLogP) for the exemplary compounds was determined using the MolecularDescriptors algorithm in the Molecular Operating Environment software(see e.g., Labute P, The Derivation and Applications of MolecularDescriptors Based Upon (Approximate) Surface Area; in Chemoinformatics:Concepts, Methods, and Tools for Drug Discovery, J. Bajorath ed. 2003).¹H NMR and mass spectrometry data are detailed below for certaincompounds of the present invention, and Table 3 provides purity data andcLogP values.

TABLE 3 (J-1)

% Purity Compound No. Formula (J-1), R⁰ (HPLC) cLogP 63-33—(CH₂)₂-cyclopropyl 95 3.9 63-35 —(CH₂)₂-cyclobutyl 97 4.4 63-36—(CH₂)₂-cyclopentyl 88 4.9 63-39 —(CH₂)-2-methylcyclopropyl 90 ND 63-40—(CH₂)-2,2-dimethylcyclo- 93 ND propyl 63-42 —(CH₂)₂-1-methylcyclopropyl90 ND 63-43 —(CH₂)₃-cyclopropyl 95 ND 63-46 —(CH₂)-3-methylcyclobutyl 93ND ND = not determined.

Compound No. 63-33: ¹H NMR (CDCl₃, 400 MHz): δ 7.80 (dd, J=8.5, 1.0 Hz,1H), 7.70 (dd, J=8.3, 1.1 Hz, 1H), 7.42 (m, J=8.4. 7.0, 1.4 Hz, 1H),7.29 (as, 1H), 7.25 (as, 1H), 7.10 (m, J=8.2, 7.1, 1.3 Hz, 1H), 7.00 (d,J=8.4 Hz, 2H), 5.92 (bs, 2H), 5.69 (s, 2H), 3.75 (s, 2H), 2.86 (dd,J=8.0 Hz, 2H), 2.68 (dd, J=8.0 Hz, 2H), 1.82-1.74 (m, 2H), 1.45-1.36 (m,4H), 0.91 (t, J=7.8 Hz, 3H), 0.91-0.85 (m, 1H), 0.68-0.60 (m, 1H),0.42-0.37 (m, 2H), 0.04-0.00 (m, 2H). Mass Spec: m/z 428.6 (M+1).

Compound No. 63-35: ¹H NMR (CDCl₃, 300 MHz): δ 7.8 (d, J=8.4 Hz, 1H),7.70 (d, J=8.5 Hz, 1H), 7.45 (t, J=8.4, 1H), 7.34 (d, J=8.5 Hz, 2H),7.10 (t, J=8.4 Hz, 1H), 7.02 (d, J=8.3 Hz, 2H), 5.72 (s, 2H), 3.85 (s,2H), 2.89 (t, J=7.6 Hz, 2H), 2.60 (t, J=7.2 Hz, 2H), 2.20-2.35 (m, 1H),1.8-2.1(m, 2H), 1.65-1.8 (m, 6H), 1.4-1.55 (m, 4H), 0.94 (t, J=7.3 Hz,3H). Mass Spec: m/z 442.9 (M+1).

Compound No. 63-36: ¹H NMR (CDCl₃, 300 MHz): δ 7.8 (d, J=8.4 Hz, 1H),7.70 (d, J=8.5 Hz, 1H), 7.45 (t, J=8.4, 1H), 7.34 (d, J=8.5 Hz, 2H),7.10 (t, J=8.4 Hz, 1H), 7.02 (d, J=8.3 Hz, 2H), 5.72 (s, 2H), 3.80 (brs, 4H), 2.89 (t, J=7.6 Hz, 2H), 2.62 (t, J=7.2 Hz, 2H), 1.62-1.84 (m,7H), 1.0-1.20 (m, 1H), 0.94 (t, J=7.3 Hz, 3H). Mass Spec: m/z 456.4(M+1).

Compound No. 63-39: ¹H NMR (CD₃OD, 300 MHz): δ 7.80 (d, J=8.1 Hz, 1H),7.67 (d, J=8.4 Hz, 1H), 7.30-7.48 (m, 3H), 7.05-7.14 (m, 3H), 5.88 (s,2H), 3.81 (s, 2H), 2.89 (t, J=7.5, 15.6 Hz, 2H), 2.44-2.55 (m, 2H),1.70-1.85 (m, 2H), 1.35-1.55 (m, 3H), 1.02 (s, J=6 Hz, 3H), 0.94 (t,J=7.5, 14.7 Hz, 3H), 0.50-0.75 (m, 2H), 0.20-0.35 (m, 2H). Mass Spec:m/z 428.4 [M+1].

Compound No. 63-40: ¹H NMR (CDCl₃, 300 MHz): δ 7.82 (d, J=8.1 Hz, 1H),7.73 (d, J=8.4 Hz, 1H), 7.45 (t, J=7.5, 15.0 Hz, 1H), 7.29 (d, J=8.4 Hz,2H), 7.15 (t, J=7.5, 15.0 Hz, 1H), 7.03 (d, J=8.4 Hz, 2H), 5.73 (s, 2H),5.54 (s, 2H), 3.78 (s, 2H), 2.50-2.70 (m, 2H), 1.75-1.80 (m, 2H),1.40-1.55 (m, 2H), 1.04 (s, 3H), 1.02 (s, 3H), 0.94 (t, J=7.5, 14.7 Hz,3H), 0.7-0.8 (m, 1H), 0.4-0.5 (m, 1H), 0.1-0.05 (m, 1H). Mass Spec: m/z442.5 [M+1].

Compound No. 63-42: ¹H NMR (CDCl₃, 300 MHz): δ 7.80 (d, J=8.1 Hz, 1H),7.71 (d, J=8.4 Hz, 1H), 7.43 (t, J=7.5, 15.0 Hz, 1H), 7.31 (d, J=8.4 Hz,2H), 7.14 (t, J=7.5, 15.0 Hz, 1H), 7.01 (d, J=8.4 Hz, 2H), 5.71 (s, 2H),3.76 (s, 2H), 2.89 (t, J=7.5, 15.6 Hz, 2H), 2.70 (t, J=7.2, 15.0 Hz,2H), 1.77-1.92 (m, 2H), 1.70-1.84 (m, 4H), 0.98 (s, 3H), 0.94 (t, J=7.5,14.7 Hz, 3H), 0.18-0.35 (m, 4H). Mass Spec: m/z 442.5 [M+1].

Compound No. 63-43: ¹H NMR (CDCl₃, 300 MHz): δ 7.80 (d, J=8.1 Hz, 1H),7.71 (d, J=8.4 Hz, 1H), 7.43 (t, J=7.5, 15.0 Hz, 1H), 7.25-7.31 (m, 2H),7.14 (t, J=7.5, 15.0 Hz, 1H), 7.01 (d, J=8.4 Hz, 2H), 5.71 (s, 2H),5.59(s, 2H), 3.75 (s, 2H), 2.88 (br t, 2H), 2.63 (t, J=7.2, 14.1 Hz,3H), 0.55-0.72 (m, 1H), 0.4 (br d, 2H), 0.01 (d, J=4.5 Hz, 2H). MassSpec: m/z 442.5 [M+1].

Compound No. 63-46: ¹H NMR (CDCl₃, 300 MHz): δ 7.81 (d, J=8.1 Hz, 1H),7.74 (d, J=8.4 Hz, 1H), 7.45 (t, J=7.5, 15.0 Hz, 1H), 7.29 (d, J=8.4 Hz,2H), 7.15 (t, J=7.5, 15.0 Hz, 1H), 7.03 (d, J=8.4 Hz, 2H), 5.73 (s, 2H),5.51 (s, 2H), 3.74 (s, 2H), 2.86 (t, J=7.5, 15.6 Hz, 2H), 2.60-2.7 (m,2H), 2.1-2.4 (m, 3H), 1.6-1.85 (m, 4H), 1.40-1.55 (m, 2H), 0.95-1.15 (m,5H), 0.94 (t, J=7.5, 14.7 Hz, 3H). Mass Spec: m/z 442.4 [M+1].

Example S5: Synthesis of2-butyl-1-(4-(((cyclopropylmethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine(Compound No. 63-38)

Part A. N,N-diisopropylcarbodiimide (272 mg, 2.15 mmol) was added to asolution of cyclopropane carboxylic acid (172 mg, 2.0 mmol) andpentafluorophenol (387 mg, 2.1 mmol) in dichloromethane (4 mL), inpresence of catalytic amounts of N,N-dimethylaminopyridine (12 mg), andstirred overnight at room temperature. This mixture was then dilutedwith ether (20 mL), the precipitated urea removed by filtration, and thefiltrate concentrated to obtain crude product. This crude product wassuspended in 1% ethyl acetate/hexane and any residual precipitated ureawas again removed by filtration. The resultant filtrate was concentratedunder reduced pressure to yield 440 mg of the desired(2,3,4,5,6-pentafluorophenyl)-cyclopropane carboxylate product.

Part B. Compound No. 63-00 (80 mg, 0.22 mmol) was added to a solution of(2,3,4,5,6-pentafluorophenyl)-cyclopropane carboxylate (61 mg, 0.24mmol) in dichloromethane (3 mL) in presence of triethylamine (45 mg,0.44 mmol), and stirred for 2 hours at room temperature. The reactionmixture was then concentrated under reduced pressure and purified byflash chromatography, eluting with 3-8% methanol/dichloromethanecontaining 1% aqueous ammonia, to obtain 78 mg ofN-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)cyclopropanecarboxamideas an off-white solid.

Part C.N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)cyclopropanecarboxamide (78 mg) was reduced with borane dimethyl sulfidecomplex (3.5 eq) at 55° C. for 12 hours. The reaction was then cooled toroom temperature, carefully quenched with 1 M HCl (excess), and stirredfor an additional 3 hours at 55° C. The reaction was cooled to roomtemperature and diluted with water (10 mL), and then extracted withdichloromethane (10 mL) to remove impurities. The pH of the reactionmixture was adjusted to 8.0 by adding an ice-cold 1M NaOH solution,extracted with dichloromethane (3×10 mL), dried over MgSO₄, andconcentrated under reduced pressure to yield an off-white solid. Uponrecrystallization with 9:1 ethyl acetate/hexane the reaction yielded 27mg of2-butyl-1-(4-(((cyclopropylmethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine(Compound No. 63-38). Product purity was assessed to be 96% pure byreverse-phase HPLC at 254 nm, the intended synthetic mass of 413.3Daltons was confirmed by LC/MS, and the intended synthetic structureconfirmed by 300 MHz ¹H NMR (CD₃OD): δ 7.80 (d, J=8.1 Hz, 1H), 7.67 (d,J=8.4 Hz, 1H), 7.38-7.48 (m, 3H), 7.07-7.20 (m, 3H), 5.92 (s, 2H), 3.97(s, 2H), 3.0 (t, J=7.8, 15.3 Hz, 2H), 2.66 (2, J=7.2 Hz, 2H), 1.78-1.90(m, 2H), 1.40-1.60 (m, 2H), 1.0 (t, J=7.5 Hz, 3H), 0.5-0.6 (m, 2H),0.2-0.3 (m, 2H).

Example S6: Synthesis of2-butyl-1-(4-((((1-methylcyclobutyl)methyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinoline-4-amine(Compound No. 63-45)

Part A. N,N-diisopropylcarbodiimide (76 mg, 0.6 mmol) was added to asolution of 1-methylcyclobutane carboxylic acid (57 mg, 0.5 mmol) andpentafluorophenol (94 mg, 0.52 mmol) in dichloromethane (3 mL), inpresence of catalytic amount of N,N-dimethylaminopyridine (6 mg), andstirred overnight at room temperature. This mixture was then dilutedwith ether (20 mL), the precipitated urea removed by filtration, and thefiltrate concentrated to obtain crude product. This crude product wassuspended in 1% ethyl acetate/hexane and any residual precipitated ureawas again removed by filtration. The resultant filtrate was concentratedunder reduced pressure to yield 126 mg of the desired(2,3,4,5,6-pentafluorophenyl)-1-methylcyclobutane carboxylate product.

Part B. Compound No. 63-00 (84 mg, 0.23 mmol) was added to a solution of(2,3,4,5,6-pentafluorophenyl)-1-methylcyclobutane carboxylate (72 mg,0.26 mmol) in dichloromethane (3 mL) in presence of triethylamine (48mg, 0.47 mmol), and stirred for 2 hours at room temperature. Thereaction mixture was then concentrated under reduced pressure and theresidue washed with 5% ethyl acetate/hexane. The residue was dissolvedin dichloromethane (15 mL), washed with 1M HCl followed by water (20mL), dried over magnesium sulfate, filtered and concentrated underreduced pressure to yield 88 mg ofN-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-1-methylcyclobutane-1-carboxamideas off white solid.

Part C.N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-1-methylcyclobutane-1-carboxamide(88 mg) was reduced with borane dimethyl sulfide complex (3.5 eq) at 55°C. for 12 hours. The reaction was then cooled to room temperature,carefully quenched with 2 M HCl (excess), and stirred for an additional3 hours at 55° C. The reaction was cooled to room temperature anddiluted with water (10 mL), then extracted with dichloromethane (10 mL)to remove impurities. The pH of the reaction mixture was adjusted to 8.0by adding an ice-cold 1M NaOH solution, extracted with dichloromethane(3×10 mL), dried over MgSO₄, and concentrated under reduced pressure toyield an off-white solid. Upon recrystallization with 9:1 ethylacetate/hexane the reaction yielded 32 mg of2-butyl-1-(4-((((1-methylcyclobutyl)methyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine(Compound No. 63-45). Product purity was assessed to be 96% pure byreverse-phase HPLC at 254 nm, the intended synthetic mass of 441.3Daltons was confirmed by LC/MS, and the intended synthetic structureconfirmed by 300 MHz ¹H NMR (CDCl₃): δ 7.80 (d, J=8.1 Hz, 1H), 7.71 (d,J=8.4 Hz, 1H), 7.43 (t, J=7.5, 15.0 Hz, 1H), 7.31 (d, J=8.4 Hz, 2H),7.14 (t, J=7.5, 15.0 Hz, 1H), 7.01 (d, J=8.4 Hz, 2H), 5.71 (s, 2H), 5.52(s, 2H), 3.78 (s, 2H), 2.89 (t, J=7.5, 15.6 Hz, 2H), 2.52 (s, 2H),1.6-1.88 (m, 8H), 1.35-1.60 (m, 2H), 1.12 (s, 3H), 0.94 (t, J=7.5, 14.7Hz, 3H).

Example S7: Synthesis of2-butyl-1-(4-(((cyclobutylmethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinoline-4-amine(Compound No. 63-44)

Part A. N,N-diisopropylcarbodiimide (127 mg, 1.0 mmol) was added to asolution of cyclobutane carboxylic acid (106 mg, 0.93 mmol) andpentafluorophenol (175 mg, 0.97 mmol) in dichloromethane (3 mL), inpresence of catalytic amount of N,N-dimethylaminopyridine (12 mg), andstirred overnight at room temperature. This mixture was then dilutedwith ether (20 mL), the precipitated urea removed by filtration, and thefiltrate concentrated to obtain crude product. This crude product wassuspended in 1% ethyl acetate/hexane and any residual precipitated ureawas again removed by filtration. The resultant filtrate was concentratedunder reduced pressure to yield 126 mg of the desired(2,3,4,5,6-pentafluorophenyl)cyclobutane carboxylate product.

Part B. Compound No. 63-00 (62 mg, 0.17 mmol) was added to a solution of(2,3,4,5,6-pentafluorophenyl)cyclobutane carboxylate (50 mg, 0.18 mmol)in dichloromethane (3 mL), in presence of triethylamine (35 mg, 0.34mmol), and stirred for 2 hours at room temperature. The reaction mixturewas then concentrated under reduced pressure and the residue washed with5% ethyl acetate/hexane. The residue was dissolved in dichloromethane(15 mL), washed with 1M HCl followed by water (20 mL), dried overmagnesium sulfate, filtered and concentrated under reduced pressure toyield 85 mg ofN-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)cyclobutanecarboxamideas an off-white solid.

Part C.N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)cyclobutanecarboxamide(85 mg) was reduced with borane dimethyl sulfide complex (3.5 eq) at 55°C. for 12 hours. The reaction was then cooled to room temperature,carefully quenched with 2 M HCl (excess), and stirred for an additional3 hours at 55° C. The reaction was cooled to room temperature anddiluted with water (10 mL), then extracted with dichloromethane (10 mL)to remove impurities. The pH of the reaction mixture was adjusted to 8.0by adding an ice-cold 1M NaOH solution, extracted with dichloromethane(3×10 mL), dried over MgSO₄, and concentrated under reduced pressure toyield an off-white solid. Upon recrystallization with 9:1 ethylacetate/hexane the reaction yielded 21 mg of2-butyl-1-(4-(((cyclobutylmethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine(Compound No. 63-44). Product purity was assessed to be 95% pure byreverse-phase HPLC at 254 nm, the intended synthetic mass of 427.3Daltons was confirmed by LC/MS, and the intended synthetic structureconfirmed by 300 MHz ¹H NMR (CDCl₃): δ 7.80 (d, J=8.1 Hz, 1H), 7.73 (d,J=8.4 Hz, 1H), 7.45 (t, J=7.5, 15.0 Hz, 1H), 7.29 (d, J=8.4 Hz, 2H),7.15 (t, J=7.5, 15.0 Hz, 1H), 7.03 (d, J=8.4 Hz, 2H), 5.72 (s, 2H), 5.51(s, 2H), 3.74 (s, 2H), 2.89 (t, J=7.5, 15.6 Hz, 2H), 2.62 (d, J=7.2 Hz,2H), 2.40-2.58 (m, 1H), 1.75-2.15 (m, 7H), 1.60-1.70 (m, 2H), 1.35-1.50(m, 2H), 0.94 (t, J=7.5, 14.7 Hz, 3H).

Example S8: Synthesis of2-butyl-1-(4-(((2-cyclobutyl-2-methylpropyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine(Compound No. 63-47)

Part A. N,N-diisopropylcarbodiimide (113 mg, 0.89 mmol) was added to asolution of 3-cyclobutyl-3-methyl-butan-2-one (70 mg, 0.5 mmol) andpentafluorophenol (108 mg, 0.59 mmol) in dichloromethane (3 mL), inpresence of catalytic amount of N,N-dimethylaminopyridine (11 mg), andstirred overnight at room temperature. This mixture was then dilutedwith ether (20 mL), the precipitated urea removed by filtration, and thefiltrate concentrated to obtain crude product. This crude product wassuspended in 1% ethyl acetate/hexane and any residual precipitated ureawas again removed by filtration. The resultant filtrate was concentratedunder reduced pressure to yield 141 mg of the desired(2,3,4,5,6-pentafluorophenyl)-2-cyclobutyl-2-methyl-propanoate product.

Part B. Compound No. 63-00 (60 mg, 0.17 mmol) was added to a solution of(2,3,4,5,6-pentafluorophenyl)-2-cyclobutyl-2-methyl-propanoate (54 mg,0.18 mmol) in dichloromethane (3 mL), in presence of triethylamine (34mg, 0.34 mmol), and stirred at reflux for 3 days. The reaction mixturewas then cooled to room temperature, concentrated under reducedpressure, and the residue washed with 5% ethyl acetate/hexane. Thewashed residue was dissolved in dichloromethane (15 mL), washed with 1MHCl followed by water (20 mL), dried over magnesium sulfate, filtered,and concentrated under reduced pressure to yield 67 mg ofN-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-2-cyclobutyl-2-methylpropanamideas an off-white solid.

Part C.N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-2-cyclobutyl-2-methylpropanamide(67 mg) was reduced with borane dimethyl sulfide complex (3.5 eq) at 55°C. for 12 hours. The reaction was then cooled to room temperature,carefully quenched with 2 M HCl (excess), and stirred for an additional3 hours at 55° C. The reaction was cooled to room temperature anddiluted with water (10 mL), and then extracted with dichloromethane (10mL) to remove impurities. The pH of the reaction mixture was adjusted to8.0 by adding an ice-cold 1M NaOH solution, extracted withdichloromethane (3×10 mL), dried over MgSO₄, and concentrated underreduced pressure to yield an off-white solid. Upon recrystallizationwith 9:1 ethyl acetate/hexane the reaction yielded 13 mg of2-butyl-1-(4-(((2-cyclobutyl-2-methylpropyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine(Compound No. 63-47). Product purity was assessed to be 92% pure byreverse-phase HPLC at 254 nm, the intended synthetic mass of 469.3Daltons was confirmed by LC/MS, and the intended synthetic structureconfirmed by 300 MHz ¹H NMR (CDCl₃): δ 7.80 (d, J=8.1 Hz, 1H), 7.71 (d,J=8.4 Hz, 1H), 7.43 (t, J=7.5, 15.0 Hz, 1H), 7.31 (d, J=8.4 Hz, 2H),7.14 (t, J=7.5, 15.0 Hz, 1H), 7.01 (d, J=8.4 Hz, 2H), 6.60 (br s, 1H),5.73 (s, 2H), 3.74 (br s, 4H), 2.90 (t, J=7.5, 15.6 Hz, 2H), 2.35 (s,2H), 1.20-1.8 (m, 10H), 0.94 (t, J=7.5, 14.7 Hz, 3H), 0.90 (s, 6H).

Example S9: Synthesis of2-butyl-1-(4-(((2-cyclopropyl-2-methylpropyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinoline-4-amine(Compound No. 63-41)

Part A. N,N-diisopropylcarbodiimide (127 mg, 1.0 mmol) was added to asolution of 2-cyclopropyl-2-methyl-propanoic acid (106 mg, 0.93 mmol)and pentafluorophenol (175 mg, 0.97 mmol) in dichloromethane (3 mL), inthe presence of catalytic amounts of N,N-dimethylaminopyridine (12 mg),and stirred overnight at room temperature. This mixture was then dilutedwith ether (20 mL), the precipitated urea removed by filtration, and thefiltrate concentrated to obtain crude product. This crude product wassuspended in 1% ethyl acetate/hexane and any residual precipitated ureawas again removed by filtration. The resultant filtrate was concentratedunder reduced pressure to yield 130 mg of the desired(2,3,4,5,6-pentafluorophenyl)-2,2-dimethylcyclopropanecarboxylateproduct.

Part B. Compound No. 63-00 (62 mg, 0.17 mmol) was added to a solution of(2,3,4,5,6-pentafluorophenyl)-2,2-dimethylcyclopropanecarboxylate (50mg, 0.18 mmol) in dichloromethane (3 mL), in presence of triethylamine(35 mg, 0.34 mmol), and stirred for 2 hours at room temperature. Thereaction mixture was then concentrated under reduced pressure and theresidue washed with 5% ethyl acetate/hexane. The washed residue wasdissolved in dichloromethane (15 mL), washed with 1M HCl followed bywater (20 mL), dried over magnesium sulfate, filtered and concentratedunder reduced pressure to yield 85 mg ofN-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-2-cyclopropyl-2-methylpropanamideas an off-white solid.

Part C.N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-2-cyclopropyl-2-methylpropanamide(85 mg) was reduced with borane dimethyl sulfide complex (3.5 eq) at 55°C. for 12 hours. The reaction was then cooled to room temperature,carefully quenched with 2 M HCl (excess), and stirred for an additional3 hours at 55° C. The reaction was cooled to room temperature anddiluted with water (10 mL), and then extracted with dichloromethane (10mL) to remove impurities. The pH of the reaction mixture was adjusted to8.0 by adding an ice-cold 1M NaOH solution, extracted withdichloromethane (3×10 mL), dried over MgSO₄, and concentrated underreduced pressure to yield an off-white solid. Upon recrystallizationwith 9:1 ethyl acetate/hexane the reaction yielded 14 mg of2-butyl-1-(4-(((2-cyclopropyl-2-methylpropyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine(Compound No. 63-41). Product purity was assessed to be 94% pure byreverse-phase HPLC at 254 nm, the intended synthetic mass of 455.3Daltons was confirmed by LC/MS, and the intended synthetic structureconfirmed by 300 MHz ¹H NMR (CDCl₃): δ 7.82 (d, J=8.1 Hz, 1H), 7.75 (d,J=8.4 Hz, 1H), 7.45 (t, J=7.5, 15.0 Hz, 1H), 7.29 (d, J=8.4 Hz, 2H),7.15 (t, J=7.5, 15.0 Hz, 1H), 7.03 (d, J=8.4 Hz, 2H), 5.72 (s, 2H), 5.62(s, 2H), 3.79 (s, 2H), 2.90 (t, J=7.5, 15.6 Hz, 2H), 2.87 (s, 2H),1.75-1.90 (m, 2), 1.40-1.55 (m, 2H), 0.94 (t, J=7.5, 14.7 Hz, 3H), 0.75(s, 6H), 0.6-0.75 (m, 1H), 0.15-0.3 (m, 4H).

Example S10: Synthesis of2-butyl-1-(4-((cyclohexylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine(Compound No. 63-49)

Part A. Tert-butyl dimethyl silyl chloride (3.31 g, 22 mmol) was addedto a solution of 4-cyanobenzyl alcohol (2.66 g, 20 mmol) inN,N-dimethylformamide (20 mL) in the presence of imidazole (2.72 g, 40mmol) at room temperature and stirred for 4 hours. The reaction mixturewas poured into water (150 mL) and extracted with 10% ethylacetate/hexane (3×75 mL). The combined organic extract was washed withwater, dried over anhydrous magnesium sulfate, and concentrated underreduced pressure. The resulting product was further purified by flashchromatography, eluting with hexane, to yield 4.84 g of4-(((tert-butyldimethylsilyl)oxy)methyl)benzonitrile.

Part B. A solution of4-(((tert-butyldimethylsilyl)oxy)methyl)benzonitrile (4.84 g) inmethanol (200 mL) was hydrogenated in a Parr hydrogenation apparatus inthe presence of Raney Nickel (1.0 g slurry in water) under Hydrogen at60 psi for 4 hours. The reaction was filtered and the filtrateconcentrated under reduced pressure. The resulting product,(4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)methanamine (4.8 g), wasused without purification.

Part C. A solution of 4-chloro-3-nitroquinoline (4.16 g, 20 mmol) indichloromethane (100 mL) was slowly added to a solution of4-(tert-butyldimethylsiloxymethyl) benzyl amine (4.8 g, 19 mmol) anddiisopropylethylamine (DIPEA) (3.87 g, 30 mmol) in dichloromethane (100mL) and stirred for 12 hours. The solvent was removed under reducedpressure, the residue dissolved in ethyl acetate (200 mL), washed withwater (2×100 mL), and dried over anhydrous magnesium sulfate. Thesolvent was removed under reduced pressure and the resulting product waspurified by flash chromatography, eluting with 1:1 ethyl acetate/hexane,to yield 4.67 g of(4-((4-(((tert-butyldimethylsilyl)oxy)methyl)benzyl)amino)quinolin-3-yl)(oxo)-λ⁴-azanol.

Part D. A solution of(4-((4-(((tert-butyldimethylsilyl)oxy)methyl)benzyl)amino)-quinolin-3-yl)(oxo)-λ⁴-azanol(4.67 g) in ethyl acetate (250 mL) was hydrogenated in the presence ofpalladium on carbon (10%, 1.0 g) at 60 psi for 4 hours. The catalyst wasremoved by filtration, the solvent removed from the filtrate underreduced pressure, and the product,N⁴-(4-(((tert-butyldimethylsilyl)oxy)methyl)benzyl)quinoline-3,4-diamine(4.5 g), used without further purification.

Part E. A solution of valeryl chloride (1.50 g, 12.46 mmol, 1.05 eq.) indichloromethane (30 mL) was added dropwise to a solution ofN⁴-(4-(((tert-butyldimethylsilyl)oxy)methyl)-benzyl)quinoline-3,4-diamine(4.5 g, 11.86 mmol) in dry pyridine (20 mL) at 0° C. After the addition,the reaction mixture was warmed to room temperature and stirred for 4hours. The solvent and pyridine were removed under reduced pressure. Theresidue was dissolved in dichloromethane (300 mL) and then washedsuccessively with water, saturated sodium bicarbonate solution (100 mL),saturated copper sulfate solution (3×50 mL), and water (100 mL), thendried over anhydrous magnesium sulfate and concentrated under reducedpressure. The product,N-(4-((4-(((tert-butyldimethylsilyl)oxy)methyl)benzyl)amino)quinolin-3-yl)pentanamide(5.5 g), was used further without purification.

Part F. A solution ofN-(4-((4-(((tert-butyldimethylsilyl)oxy)methyl)benzyl)amino)-quinolin-3-yl)pentanamide(5.5 g, 11.87 mmol) in a mixture of ethanol/water (8:2 v/v, 150 mL) washeated to 60° C. in presence of potassium carbonate (2.5 g, 18.11 mmol,1.5 eq.) for 18 hours. The solvent was removed under reduced pressure.The residue was partitioned between ethyl acetate (200 mL) and water(100 mL), the ethyl acetate layer separated and dried over anhydrousmagnesium sulfate, and the residue concentrated under reduced pressure.Further purification was done by flash chromatography, eluting with 20%ethyl acetate/hexane, to yield 3.43 g of2-butyl-1-(4-(((tert-butyldimethylsilyl)oxy)methyl)benzyl)-1H-imidazo[4,5-c]quinoline.

Part G. Meta-chloroperbenzoic acid (60-70%, 2.5 g) was added to asolution of2-butyl-1-(4-(((tert-butyldimethylsilyl)oxy)methyl)benzyl)-1H-imidazo[4,5-c]quinoline(3.43 g, 7.4 mmol) in dichloromethane (200 mL) at room temperature andstirred for 6 hours. The reaction was quenched by adding a saturatedsolution of sodium sulfite solution (20 mL). The organic layer wasseparated, successively washed with a solution of saturated sodiumbicarbonate solution (50 mL) then water (50 mL), dried over anhydrousmagnesium sulfate and concentrated under reduced pressure. Furtherpurification was done by flash chromatography with elution by 25% ethylacetate/hexane to yield 3.1 g of2-butyl-1-(4-(((tert-butyldimethylsilyl)oxy)methyl)-benzyl)-1H-5λ⁴-imidazo[4,5-c]quinolin-5-ol.

Part H. A solution of2-butyl-1-(4-(((tert-butyldimethylsilyl)oxy)methyl)benzyl)-1H-5λ⁴-imidazo[4,5-c]quinolin-5-ol(3.1 g, 6.5 mmol) in dichloromethane (50 mL) was added to tri-n-butylamine (2.4 g, 13 mmol) and phthalimide (1.91 g, 13 mmol) and thereaction mixture was cooled to 0° C. A solution of benzoyl chloride(1.82 g, 13 mmol) in dichloromethane (10 mL) was added slowly to thereaction, and the mixture was warmed to room temperature and stirred for30 minutes. The reaction was diluted with dichloromethane (100 mL),successively washed with a saturated aqueous solution of ammoniumchloride (100 mL) then water (100 mL), and dried over anhydrousmagnesium sulfate and concentrated under reduced pressure. The resultingproduct was isolated by flash chromatography, eluting with 10% ethylacetate/hexane, to yield 2.85 g of2-(2-butyl-1-(4-(((tert-butyldimethylsilyl)oxy)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)isoindoline-1,3-dione.

Part I. A 1M solution of tetrabutylammonium fluoride (6 mL) was added toa solution of2-(2-butyl-1-(4-(((tert-butyldimethylsilyl)oxy)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)isoindoline-1,3-dione(2.85 g, 4.7 mmol) in dry tetrahydrofuran (10 mL) at 0° C. After theaddition, the reaction mixture was warmed to room temperature andfurther stirred for 6 hours. The reaction was quenched by the additionof saturated ammonium chloride (20 mL), diluted with ethyl acetate (100mL), washed with water, dried over anhydrous magnesium sulfate, andconcentrated under reduced pressure. Further purification was done byflash chromatography eluting with dichloromethane/hexane (1:1) to yield1.57 g of2-(2-butyl-1-(4-(hydroxymethyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)isoindoline-1,3-dione.

Part J. A solution of DMSO (2.5 g, 32 mmol) in CH₂Cl₂ (10 mL) was addedto a solution of oxalyl chloride (2.0 g, 16 mmol) in dichloromethane (10mL) and 3 Å molecular sieves in CH₂Cl₂ (20 mL) at −78° C. under argon.After 15 minutes, a solution of2-(2-butyl-1-(4-(hydroxymethyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)isoindoline-1,3-dione(1.57 g, 3.2 mmol) in CH₂Cl₂ (3 mL) was slowly added dropwise. After 30min, Et₃N (4.5 g, 45 mmol) was added dropwise, the reaction stirred for30 minutes at −78° C., and then slowly allowed to warm to roomtemperature. After stirring for another hour at room temperature, thereaction mixture was quenched by the addition of saturated ammoniumchloride solution. The organic layer was separated, washed with water(25 mL), dried over anhydrous magnesium sulfate and concentrated underreduced pressure. Further purification was done by flash chromatographyto yield 1.15 g of4-((2-butyl-4-(1,3-dioxoisoindolin-2-yl)-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzaldehyde.

Part K. A solution of4-((2-butyl-4-(1,3-dioxoisoindolin-2-yl)-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzaldehyde(2.33 mmol) and cyclohexylamine (7 mmol) in dichloromethane is heated toreflux in the presence of a catalytic amount of p-toluenesulfonic acidfor 12 hours. The reaction mixture is concentrated under reducedpressure. The crude product,2-(2-butyl-1-(4((cyclohexylimino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)isoindoline-1,3-dione,is used without further purification.

Part L. Sodium borohydride (10 mmol) is added to a solution of2-(2-butyl-1-(4-(cyclohexylimino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)isoindoline-1,3-dionein methanol at room temperature and stirred for 2 hours. The reactionmixture is quenched with saturated ammonium chloride solution andextracted with dichloromethane (3×30 mL). The combined organic layer iswashed with water, dried over anhydrous magnesium sulfate andconcentrated under reduced pressure. The product,2-(2-butyl-1-(4-((cyclohexylamino)methyl)-benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)isoindoline-1,3-dione,is purified by flash chromatography.

Part M. Hydrazine (100 mg) is added to a solution of2-(2-butyl-1-(4-((cyclohexylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)isoindoline-1,3-dione(0.38 mmol) in methanol and stirred for 12 hours. The solvent is removedunder reduced pressure and the resulting product,2-butyl-1-(4-((cyclohexylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine,is purified by flash chromatography.

Example S11: Synthesis of Exemplary N-Acyl Compounds of Formula (J-2)

Exemplary N-acyl compounds of formula (J-2) were synthesized usingsimilar procedures as for Compound No. 63-33, through part I. ¹H NMR andmass spectrometry data are detailed below for certain compounds of thepresent invention, and Table 4 provides purity data and cLogP values.

TABLE 4

(J-2) Compound No. Formula (J-2), R⁰ % Purity (HPLC) cLogP 63-34—(CH₂)-cyclopropyl 60 4.8

Compound No. 63-34: ¹H NMR (CDCl₃, 400 MHz): δ 7.75 (dd, J=8.3, 0.9 Hz,1H), 7.67 (dd, J=8.3, 1.1 Hz, 1H), 7.40 (m, J=8.3, 7.1, 1.4 Hz, 1H),7.22 (d, J=8.2 Hz, 2H), 7.11 (m, J=8.2, 7.1, 1.2 Hz, 1H), 6.98 (d, J=8.2Hz, 2H), 6.30 (at, 1H), 5.68 (s, 2H), 4.42 (d, J=6.0 Hz, 2H), 2.84 (add,2H), 2.16 (dd, J=7.2 Hz, 2H), 1.81-1.75 (m, 2H), 1.48-1.37 (m, 2H), 0.91(t, J=7.4 Hz, 3H), 0.57-0.53 (m, 2H), 0.17-0.14 (m, 2H). Mass Spec: m/z442.6 (M+1).

Example S12: Synthesis of Exemplary Compounds of Formula (K)

Exemplary N-acyl compounds of formula (K-2) were synthesized usingsimilar procedures as for Compound No. 63-10. ¹H NMR and massspectrometry data are detailed below for certain compounds of thepresent invention, and Table 5 lists the compounds with purity data aswell as cLogP values.

Exemplary N-alkyl compounds of formula (K-1) were synthesized usingsimilar procedures as for Compound No. 63-17. ¹H NMR and massspectrometry data are detailed below for certain compounds of thepresent invention, and Table 6 provides purity data and cLogP values.

Compound No. 63-02: ¹H NMR (CDCl₃, 400 MHz): δ 7.80 (dd, J=8.4, 1.2 Hz,1H), 7.64 (dd, J=8.2, 1.0 Hz, 1H), 7.40 (m, J=8.4, 7.0, 1.0 Hz, 1H),7.20 (d, J=8.2 Hz, 2H), 7.09 (m, J=8.2, 7.1, 1.3 Hz, 1H), 6.96 (d, J=8.2Hz, 2H), 6.28 (bs, 2H), 6.02 (bt, J=5.7 Hz, 1H), 5.65 (s, 2H), 4.37 (d,J=5.8 Hz, 2H), 2.83 (dd, J=7.8, 7.8 Hz, 2H), 2.15 (dd, J=7.6, 7.6 Hz,2H), 1.81-1.73 (m, 2H), 1.64-1.56 (m, 2H), 1.42 (dq, J=15.0, 7.4 Hz,2H), 1.32-1.21 (m, 4H), 0.91 (t, J=7.3 Hz, 3H), 0.84 (t, J=7.3 Hz, 3H).Mass Spec: m/z 468.2 (M+1).

Compound No. 63-05: ¹H NMR (CDCl₃, 300 MHz): 7.89 (d, J=8.1 Hz, 1H),7.86 (d, J=8.1 Hz, 1H), 7.69(t, 1H), 7.74 (t, J=15.6, 8.4, 1H), 7.47 (t,J=15.5, 7.5 Hz, 1H), 7.38 (m, 4H), 7.14(bs, 1H) 7.08 (d, J=8.1 Hz) 5.85(s, 2H), 4.53 (d, J=6 Hz, 2H), 2.99 (t, J=7.8 Hz, 2H), 2.30 (t, J=7.5Hz, 2H), 1.90-1.97 (m, 4H), 1.52-1.75 (m, 4H), 1.26-1.48 (m, 14 H),0.90-1.10 (m, 6H). Mass Spec: m/z 500.8 (M+1).

Compound No. 63-06: ¹H NMR (CDCl₃, 300 MHz): 8.05 (d, J=8.1 Hz, 1H),7.84(d, J=8.4 Hz, 1H), 7.74 (t, J=8.4 Hz, 1H), 7.47 (t, J=7.5 Hz, 1H),7.38 (d, J=8.1 Hz, 2H), 7.14(d, J=8.1 Hz, 2H), 6.04 (s, 2H), 4.42 (s,2H), 3.74 (s, 2H)), 3.10 (t, J=7.8 Hz, 2H), 2.29 (t, J=7.5 Hz, 2H),1.90-1.97 (m, 2H), 1.52-1.75 (m, 4H), 1.26-1.48 (m, 12 H), 0.90-1.10 (m,6H). Mass Spec: m/z 514.5 (M+1).

Compound No. 63-07: ¹H NMR (CDCl₃, 300 MHz): 7.88 (d, J=8.1 Hz, 1H),7.69(d, J=8.4 Hz, 1H), 7.50 (t, J=15.6, 8.4, 1H), 7.29 (d, J=8.1 Hz,2H), 7.20 (t, J=15.5, 7.5 Hz, 1H), 7.06(d, J=8.1 Hz, 2H), 5.90 (s, 2H),4.33 (s, 2H), 3.74 (s, 2H)), 3.0 (t, J=15.0, 7.8 Hz, 2H), 2.21 (t,J=14.7, 7.5 Hz, 2H), 1.90-1.97 (m, 2H), 1.42-1.70 (m, 4H), 1.26-1.48 (m,14 H), 0.85-1.05 (m, 6H). Mass Spec: m/z 528.6 (M+1).

Compound No. 63-08: ¹H NMR (CDCl₃, 400 MHz): δ 7.74 (d, J=8.2 Hz, 1H),7.64 (d, J=8.2 Hz, 1H), 7.37 (dd, J=7.4, 7.4 Hz, 1H), 7.18 (d, J=8.0 Hz,2H), 7.07 (dd, J=7.5, 7.5 Hz, 1H), 6.95 (d, J=8.0 Hz, 2H), 5.98 (bt,J=5.6 Hz, 1H), 5.89 (bs, 2H), 5.63 (s, 2H), 4.35 (d, J=5.8 Hz, 2H), 2.81(dd, J=8.0, 8.0 Hz, 2H), 2.14 (dd, J=7.6, 7.6 Hz, 2H), 1.80-1.71 (m,2H), 1.61-1.56 (m, 2H), 1.39 (dq, J=15.0, 7.4 Hz, 2H), 1.32-1.21 (m,18H), 0.90 (t, J=7.3 Hz, 3H), 0.86 (t, J=7.3 Hz, 3H). Mass Spec: m/z542.4 (M+1).

Compound No. 63-09: ¹H NMR (DMSO-d6, 300 MHz): 8.18 (t, 1H), 7.8 (d,J=8.4 Hz, 1H), 7.6 (d, J=8.5 Hz, 1H), 7.34 (t, J=8.4, 1H), 7.18 (d,J=8.5 Hz, 2H), 6.9-7.12 (m, 3H), 6.55 (s, 2H), 5.8 (s, 2H), 4.2 (d, 2H),2.90 (t, J=7.6 Hz, 2H), 2.2 (t, J=7.2 Hz, 2H), 1.65-1.80 (m, 2H),1.3-1.75 (m, 4H), 1.25-1.38 (m, 10 H), 0.65-1.0 (m, 6H). Mass Spec: m/z556.9 (M+1).

Compound No. 63-10: ¹H NMR (CDCl₃, 300 MHz): 7.98 (d, J=8.1 Hz, 1H),7.75 (d, J=8.4 Hz, 1H), 7.67 (t, J=8.4 Hz, 1H), 7.40 (t, J=7.5 Hz, 1H),7.30 (d, J=8.1 Hz, 2H), 7.06 (d, J=8.1 Hz, 2H), 5.95 (s, 2H), 4.33 (s,2H), 3.74 (s, 2H)), 3.01 (t, J=7.8 Hz, 2H), 2.20 (t, J=7.5 Hz, 2H),1.82-1.9 (m, 2H), 1.42-1.70 (m, 4H), 1.26-1.48 (m, 20 H), 0.85-1.05 (m,6H). Mass Spec: m/z 570.8 (M+1).

Compound No. 63-11: ¹H NMR (CDCl₃, 300 MHz): 7.98 (d, J=8.1 Hz, 1H),7.76(d, J=8.4 Hz, 1H), 7.65 (t, J=8.4 Hz, 1H), 7.38 (t, J=7.5 Hz, 1H),7.30 (d, J=8.1 Hz, 2H), 7.06 (d, J=8.1 Hz, 2H), 5.95 (s, 2H), 4.33 (s,2H), 3.74 (s, 2H), 3.03 (t, J=7.8 Hz, 2H), 2.21 (t, J=7.5 Hz, 2H),1.82-1.9 (m, 2H), 1.42-1.70 (m, 4H), 1.26-1.48 (m, 22 H), 0.85-1.05 (m,6H). Mass Spec: m/z 585.2 (M+1).

Compound No. 63-31: ¹H NMR (CD₃OD, 300 MHz): 7.82 (d, J=8.4 Hz, 1H),7.70 (d, J=8.5 Hz, 1H), 7.42 (t, J=8.4, 1H), 7.34 (d, J=8.5 Hz, 2H),7.10 (t, J=8.4 Hz, 1H), 7.02 (d, J=8.3 Hz, 2H), 5.9 (s, 2H), 4.39 (s,2H)), 3.0 (t, J=7.6 Hz, 2H), 2.25 (t, J=7.2 Hz, 2H), 1.42-1.90 (m, 6H),1.2-1.4 (m, 13H), 0.65-1.0 (m, 6H). Mass Spec: m/z 598.7 (M+1).

Compound No. 63-13: ¹H NMR (CDCl₃, 400 MHz): δ 7.77 (d, J=8.2 Hz, 1H),7.67 (d, J=8.2 Hz, 1H), 7.41 (dd, J=7.4, 7.4 Hz, 1H), 7.21 (d, J=8.0 Hz,2H), 7.11 (dd, J=7.5, 7.5 Hz, 1H), 6.98 (d, J=8.0 Hz, 2H), 5.87 (bt,J=5.6 Hz, 1H), 5.87 (bs, 2H), 5.68 (s, 2H), 4.39 (d, J=5.8 Hz, 2H), 2.85(dd, J=8.0, 8.0 Hz, 2H), 2.17 (dd, J=7.6, 7.6 Hz, 2H), 1.83-1.75 (m,2H), 1.65-1.58 (m, 2H), 1.43 (dq, J=15.0, 7.4 Hz, 2H), 1.32-1.21 (m,30H), 0.92 (t, J=7.3 Hz, 3H), 0.88 (t, J=7.3 Hz, 3H). Mass Spec: m/z625.5 (M+1).

Compound No. 63-00: ¹H NMR (MeOHd4, 400 MHz): δ 7.70 (dd, J=8.4, 1.0 Hz,1H), 7.63 (dd, J=8.4, 0.8 Hz, 1H), 7.36 (m, J=8.4, 7.0, 1.4 Hz, 1H),7.27 (d, J=8.2 Hz, 2H), 7.03 (m, J=8.3, 7.0, 1.3 Hz, 1H), 6.97 (d, J=8.4Hz, 2H), 5.72 (s, 2H), 3.72 (s, 2H), 2.88 (dd, J=7.6, 7.6 Hz, 2H),1.77-1.69 (m, 2H), 1.45-1.35 (m, 2H), 0.90 (t, J=7.4 Hz, 3H). Mass Spec:m/z 360.2 (M+1).

Compound No. 63-17: ¹H NMR (CDCl₃, 300 MHz): 7.8 (d, J=8.4 Hz, 1H), 7.70(d, J=8.5 Hz, 1H), 7.45 (t, J=8.4, 1H), 7.34 (d, J=8.5 Hz, 2H), 7.10 (t,J=8.4 Hz, 1H), 7.02 (d, J=8.3 Hz, 2H), 5.70 (br s, 4H), 3.80 (s, 2H)),2.89 (t, J=7.6 Hz, 2H), 2.62 (t, J=7.2 Hz, 2H), 1.75-1.84 (m, 2H),1.2-1.75 (m, 8H), 0.65-1.0 (m, 6H). Mass Spec: m/z 430.3 (M+1).

Compound No. 63-18: ¹H NMR (CDCl₃, 300 MHz): 7.8 (d, J=8.4 Hz, 1H), 7.70(d, J=8.5 Hz, 1H), 7.45 (t, J=8.4, 1H), 7.34 (d, J=8.5 Hz, 2H), 7.10 (t,J=8.4 Hz, 1H), 7.02 (d, J=8.3 Hz, 2H), 5.72 (br s, 4H), 3.76 (s, 2H)),2.89 (t, J=7.6 Hz, 2H), 2.62 (t, J=7.2 Hz, 2H), 1.75-1.84 (m, 2H),1.4-1.6 (m, 4H), 1.2-1.35 (br s, 4H), 0.65-1.0 (m, 6H). Mass Spec: m/z444.6 (M+1).

Compound No. 63-19: ¹H NMR (CDCl₃, 300 MHz): 7.8 (d, J=8.4 Hz, 1H), 7.70(d, J=8.5 Hz, 1H), 7.45 (t, J=8.4, 1H), 7.34 (d, J=8.5 Hz, 2H), 7.10 (t,J=8.4 Hz, 1H), 7.02 (d, J=8.3 Hz, 2H), 5.70 (br s, 4H), 3.80 (s, 2H)),2.89 (t, J=7.6 Hz, 2H), 2.62 (t, J=7.2 Hz, 2H), 1.75-1.84 (m, 2H),1.4-1.6 (m, 4H), 1.2-1.35 (b, s, 8H), 0.65-1.0 (m, 6H). Mass Spec: m/z458.4 (M+1).

Compound No. 63-20: ¹H NMR (CDCl₃, 300 MHz): 7.8 (d, J=8.4 Hz, 1H), 7.70(d, J=8.5 Hz, 1H), 7.45 (t, J=8.4, 1H), 7.34 (d, J=8.5 Hz, 2H), 7.10 (t,J=8.4 Hz, 1H), 7.02 (d, J=8.3 Hz, 2H), 5.70 (br s, 4H), 3.80 (s, 2H)),2.89 (t, J=7.6 Hz, 2H), 2.62 (t, J=7.2 Hz, 2H), 1.75-1.84 (m, 2H),1.4-1.6 (m, 4H), 1.2-1.35 (b, s, 10H), 0.65-1.0 (m, 6H). Mass Spec: m/z472.4 (M+1).

Compound No. 63-21: ¹H NMR (CDCl₃, 300 MHz): 7.8 (d, J=8.4 Hz, 1H), 7.70(d, J=8.5 Hz, 1H), 7.45 (t, J=8.4, 1H), 7.34 (d, J=8.5 Hz, 2H), 7.10 (t,J=8.4 Hz, 1H), 7.02 (d, J=8.3 Hz, 2H), 5.70 (br s, 4H), 3.80 (s, 2H)),2.89 (t, J=7.6 Hz, 2H), 2.62 (t, J=7.2 Hz, 2H), 1.75-1.84 (m, 2H),1.4-1.6 (m, 4H), 1.2-1.35 (b, s, 12H), 0.65-1.0 (m, 6H). Mass Spec: m/z486.5 (M+1).

Compound No. 63-22: ¹H NMR (CDCl₃, 300 MHz): 7.8 (d, J=8.4 Hz, 1H), 7.70(d, J=8.5 Hz, 1H), 7.45 (t, J=8.4, 1H), 7.34 (d, J=8.5 Hz, 2H), 7.10 (t,J=8.4 Hz, 1H), 7.02 (d, J=8.3 Hz, 2H), 5.70 (br s, 4H), 3.80 (s, 2H)),2.89 (t, J=7.6 Hz, 2H), 2.62 (t, J=7.2 Hz, 2H), 1.75-1.84 (m, 2H),1.4-1.6 (m, 4H), 1.2-1.35 (b, s, 14H), 0.65-1.0 (m, 6H). Mass Spec: m/z500.5 (M+1).

Compound No. 63-24: ¹H NMR (CDCl₃, 300 MHz): 7.78 (d, J=8.4 Hz, 1H),7.70 (d, J=8.5 Hz, 1H), 7.43 (t, J=8.4, 1H), 7.28 (d, J=8.5 Hz, 2H),7.10 (t, J=8.4 Hz, 1H), 6.98 (d, J=8.3 Hz, 2H), 5.70 (br s, 4H), 3.73(s, 2H)), 2.87 (t, J=7.6 Hz, 2H), 2.58 (t, J=7.2 Hz, 2H), 1.71-1.84 (m,2H), 1.38-1.55 (m, 4H), 1.10-1.35 (m, 18H), 0.65-1.0 (m, 6H). Mass Spec:m/z 528.7 (M+1).

Compound No. 63-32: ¹H NMR (CDCl₃, 300 MHz): 7.8 (d, J=8.4 Hz, 1H), 7.73(d, J=8.5 Hz, 1H), 7.42 (t, J=8.4, 1H), 7.34 (d, J=8.5 Hz, 2H), 7.12 (t,J=8.4 Hz, 1H), 7.02 (d, J=8.3 Hz, 2H), 5.73 (s, 2H), 5.5 (br s, 2H) 3.75(s, 2H)), 2.90 (t, J=7.6 Hz, 2H), 2.62 (t, J=7.2 Hz, 2H), 1.75-1.85 (m,4H), 1.4-1.6 (m, 4H), 1.25-1.38 (m, 15 H), 0.65-1.0 (m, 6H). Mass Spec:m/z 584.9 (M+1).

Compound No. 63-29: ¹H NMR (CDCl₃, 300 MHz): 7.8 (d, J=8.4 Hz, 1H), 7.73(d, J=8.5 Hz, 1H), 7.42 (t, J=8.4, 1H), 7.34 (d, J=8.5 Hz, 2H), 7.12 (t,J=8.4 Hz, 1H), 7.02 (d, J=8.3 Hz, 2H), 5.72 (s, 2H), 3.80 (s, 2H), 3.78(s, 2H), 2.90 (t, J=7.6 Hz, 2H), 2.62 (t, J=7.2 Hz, 2H), 1.65-1.85 (m,2H), 1.35-1.6 (m, 4H), 1.25-1.38 (m, 15 H), 0.65-1.0 (m, 6H). Mass Spec:m/z 612.8 (M+1).

TABLE 5

(K-2) Compound No. Formula (K-2), R % Purity (HPLC) cLogP 63-02 —C₅H₁₁91 4.6 63-05 —C₈H₁₇ 98 6.1 63-06 —C₉H₁₉ 95 6.6 63-07 —C₁₀H₂₁ 94 7.263-08 —C₁₁H₂₃ 96 7.7 63-09 —C₁₂H₂₅ 99 8.2 63-10 —C₁₃H₂₇ 96 8.7 63-11—C₁₄H₂₉ 94 9.2 63-13 —C₁₇H₃₅ 92 10.7  Compound No. Comparative Compound,R % Purity (HPLC) cLogP 63-31 —C₁₅H₃₁ 95 9.7

TABLE 6

(K-1) Compound No. Formula (K-1), R″ % Purity (HPLC) cLogP 63-17 —C₅H₁₁90 0.7 63-18 —C₆H₁₃ 91 1.2 63-19 —C₇H₁₅ 90 1.7 63-20 —C₈H₁₇ 92 2.2 63-21—C₉H₁₉ 91 2.7 63-22 —C₁₀H₂₁ 93 3.2 63-24 —C₁₂H₂₅ 92 4.2 63-29 —C₁₈H₃₇ 937.2 Compound No. Comparative Compound, R″ % Purity (HPLC) cLogP 63-00 —H98 −1.1   63-32 —C₁₆H₃₃ 93 6.2

Example S13: Synthesis of Additional Exemplary N-Alkyl Compounds ofFormula (K-1)

The exemplary N-alkyl Compound Nos. 63-16, 63-23, 63-25, 63-26, 63-27,63-28, and 63-30 of formula (K-1) are synthesized using a similarprocedure as described for Compound No. 63-17 (Example S2). In Part I,the following carboxylic acids are used in place of the valeric acidused for Compound No. 63-17: n-butanoic acid (Compound No. 63-16);undecanoic acid (Compound No. 63-23); tridecanoic acid (Compound No.63-25); tetradecanoic acid (Compound No. 63-26); pentadecanoic acid(Compound No. 63-27); heptadecanoic acid (Compound No. 63-28); andnonadecanoic acid (Compound No. 63-30). The resulting N-acyl derivativesare then reduced to the final N-alkyl compounds according to Part Jdescribed for Example S2.

Example S14: Synthesis of Additional Exemplary N-Acyl Compounds ofFormula (K-2)

The exemplary N-acyl Compound Nos. 63-01, 63-03, 63-04, 63-12, 63-14,and 63-15 of formula (K-2) are synthesized using a similar procedure asdescribed for Compound No. 63-10 (Example S1) through Part H. In Part I,the following carboxylic acids are used in place of the myristic acidused for Compound No. 63-10: pentanoic acid (Compound No. 63-01),heptanoic acid (Compound No. 63-03), octanoic acid (Compound No. 63-04),heptadecanoic acid (Compound No. 63-12), nonadecanoic acid (Compound No.63-14), and arachidic acid (Compound No. 63-15), to form the N-acylproducts.

Biological Examples Example B1. In Vitro Biological Assays and Results

Methods

Plasmacytoid dendritic cell (pDC)—enriched peripheral blood mononuclearcells (PBMCs) were prepared from the blood of a series of human donors(3-5 donors/experiment). PBMCs were isolated using Ficoll-Paque Premium®(GE Healthcare, Chicago Ill.) using methods well known to those in theart. pDCs were magnetically isolated from the total recovered PBMCpopulation using CD304 (BDCA-4/Neuropilin-1) microbeads (MiltenyiBiotec, San Diego Calif.), according to the manufacturer's instructions.Isolated pDCs were then added back to between 1 and 2×10⁸ of thecorresponding donor's PBMCs for relative enrichment of this cell type.Duplicate cultures of pDC-enriched PBMCs (2.5×10⁶ cells/mL in RPMI-1640media plus 10% fetal bovine serum, cultured in 96 well plates) were thenincubated for 24 hours with Compound Nos. 63-02, 63-05 through 63-11,63-13, 63-31, 63-34, 63-38 through 63-47, and their unmodified congener1-(4-aminomethylbenzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine(Compound No. 63-00) at 10 serially diluted concentrations covering therange of 0.1 nM to 400 nM. Culture supernatants were collected andinterferon-alpha (IFNα) protein levels were measured by ELISA (MabTech,Cincinnati Ohio), according to the manufacturer's instructions.

Monocytes were magnetically isolated from PBMCs, prepared as describedabove, following labeling with CD14 microbeads (Miltenyi Biotec, SanDiego Calif.), according to the manufacturer's instructions. Duplicatecultures of monocytes (1×10⁶ cells/mL in RPMI-1640 plus 10% fetal bovineserum, cultured in 96 well plates) were incubated for 24 hours withCompound Nos. 63-02, 63-05 through 63-11, 63-13, 63-31, 63-34, 63-38through 63-47, and their unmodified congener1-(4-aminomethylbenzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine(Compound No. 63-00) at 10 serially diluted concentrations covering therange of 2 nM to 40 μM. Culture supernatants were collected and tumornecrosis factor alpha (TNFα) protein levels were measured by ELISA(MabTech, Cincinnati Ohio), according to the manufacturer'sinstructions.

Results

The effect of modifying either i) the compound of formula (K-2), where Ris a linear alkyl chain of 5 (Compound No. 63-02), 8 (Compound No.63-05), 9 (Compound No. 63-06), 10 (Compound No. 63-07), 11 (CompoundNo. 63-08), 12 (Compound No. 63-09), 13 (Compound No. 63-10), 14(Compound No. 63-11), 15 (Compound No. 63-31), or 17 (Compound No.63-13) carbons added to the amide group on the benzyl methyl moiety, orii) the compound of formula (J-2), where R⁰ is a cyclopropylmethylmoiety with 4 (Compound No. 63-34) carbons added to the amide group onthe benzyl methyl moiety, on in vitro TLR7 (induction of IFNα protein inpDC-enriched PBMC cultures) and TLR8 (induction of TNFα protein inmonocyte cultures) agonist bioactivity was assessed. Table B1-1summarizes the structural, cLogP calculation, and TLR7/8 agonistbioactivity relationships for Compound Nos. 63-00, 63-02, 63-05 through63-11, 63-13, 63-31, and 63-34. The TLR7 and TLR8 agonist potency isshown in Table B1-1 and is reported as the effective concentration at50% of the maximal response, in nanomolar (EC₅₀ in nM). Modification ofCompound No. 63-00 with these alkyl chains generally resulted in a 2-3×loss of TLR7 agonist potency, except for the 5 (Compound No. 63-02) and17 (Compound No. 63-13) carbon variants which lost 11× and 41× potency,respectively. In contrast, the 5 (Compound No. 63-02), 8 (Compound No.63-05), 9 (Compound No. 63-06), 10 (Compound No. 63-07), 15 (CompoundNo. 63-31), and 17 (Compound No. 63-13) carbon chain variantslost >10-26× of TLR8 agonist potency compared to unmodified Compound No.63-00. Unexpectedly, variants with carbon chain lengths of 11 (CompoundNo. 63-08), 12 (Compound No. 63-09), 13 (Compound No. 63-10), and 14(Compound No. 63-11) did not lose as much agonist activity, withCompound No. 63-10 demonstrating only a 2.4x loss of TLR8 agonistpotency compared to unmodified Compound No. 63-00. Compound No. 63-34,where R⁰ is a cyclopropylmethyl group, demonstrated an 8-fold loss ofTLR7 agonist potency and a 7-fold loss of TLR8 agonist potency comparedto unmodified Compound No. 63-00. These data are comparable to the TLR7and TLR8 agonist potency loss observed for the linear 5 carbon alkylchain modified Compound No. 63-02.

TABLE B1-1 Structural, cLogP Calculation, and Bioactivity Relationshipsfor Select Compounds of Formula (J-2) and (K-2). TLR7 TLR8 Compound No.cLogP (EC₅₀ in nM) (EC₅₀ in nM) 63-02 4.6 11 2,137 63-05 6.1 3 2,16763-06 6.6 2 2,746 63-07 7.2 1 2,644 63-08 7.7 3 1,271 63-09 8.2 2 97063-10 8.7 2 522 63-11 9.2 2 827 63-13 10.7 41 5,723 63-34 4.8 8 1,48663-00 −1.1 1 217 63-31 9.7 3 2,152

The effect of either i) modifying the compound of formula (K-1), where Ris a linear alkyl chain of 5 (Compound No. 63-17), 6 (Compound No.63-18), 7 (Compound No. 63-19), 8 (Compound No. 63-20), 9 (Compound No.63-21), 10 (Compound No. 63-22), 12 (Compound No. 63-24), 16 (CompoundNo. 63-32), or 18 (Compound No. 63-29) carbons added to the amine groupon the benzyl methyl moiety, or ii) modifying the compound of formula(J-1), where R⁰ is a cyclopropylethyl moiety with 5 (Compound No.63-33), cyclobutylethyl moiety with 6 (Compound No. 63-35), orcyclopentylethyl moiety with 7 (Compound No. 63-36) carbons added to theamide group on in vitro TLR7 (induction of IFNα protein in pDC-enrichedPBMC cultures) and TLR8 (induction of TNFα protein in monocyte cultures)agonist bioactivity was assessed. Table B1-2 summarizes the structural,cLogP calculation, and TLR7/8 agonist bioactivity relationships forCompound Nos. 63-00, 63-17 through 63-22, 63-24, 63-29, 63-32, 63-33,63-35, and 63-36. The TLR7 and TLR8 agonist potency is shown in TableB1-2 and is reported as the effective concentration at 50% of themaximal response, in nanomolar (EC₅₀ in nM). Modification of CompoundNo. 63-00 with linear alkyl chains demonstrated increasing loss ofTLR7/8 agonist potency with increasing number of carbons, although the 5(Compound No. 63-17) and 6 (Compound No. 63-18) carbon variantsdemonstrated slightly improved TLR8 agonist potency. Modification ofCompound No. 63-00 with three hydrocarbyl groups, cyclopropylethyl,cyclobutylethyl, and cyclopentylethyl, caused a 4- to 12-fold loss ofTLR7 agonist bioactivity, but resulted in a 2-3 fold increase in agonistpotency.

TABLE B1-2 Structural, cLogP Calculation, and Bioactivity Relationshipsfor Select Compounds of Formula (J-1) and (K-1). TLR7 TLR8 Compound No.cLogP (EC₅₀ in nM) (EC₅₀ in nM) 63-17 0.7 2 104 63-18 1.2 15 213 63-191.7 24 752 63-20 2.2 51 1,221 63-21 2.7 68 947 63-22 3.2 62 821 63-244.2 32 1,036 63-29 7.2 19 2,774 63-33 3.9 4 75 63-35 4.4 12 70 63-36 4.911 117 63-00 −1.1 1 217 63-32 6.2 157 3,099

The effect of modifying the compound of formula (J-1), where R⁰ is a(cyclopropyl)ethyl moiety (Compound No. 63-33), (cyclobutyl)ethyl moiety(Compound No. 63-35), (cyclopentyl)ethyl moiety (Compound No. 63-36),(cyclopropyl)methyl moiety (Compound No. 63-38),(2-methylcyclopropyl)methyl moiety (Compound No. 63-39),(2,2-dimethylcyclopropyl)methyl moiety (Compound No. 63-40),(2-cyclopropyl)-(2,2-dimethyl)ethyl moiety (Compound No. 63-41),(1-methylcyclopropyl)ethyl moiety (Compound No. 63-42),(3-cyclopropyl)propyl moiety (Compound No. 63-43), (cyclobutyl)methylmoiety (Compound No. 63-44), (1-methylcyclobutyl)methyl moiety (CompoundNo. 63-45), (3-methylcyclobutyl)methyl moiety (Compound No. 63-46), or(2-cyclobutyl)-(2,2-dimethyl)ethyl moiety (Compound No. 63-47), on invitro TLR7 (induction of IFNα protein in pDC-enriched human PBMCcultures) and TLR8 (induction of TNFα protein in human monocytecultures) agonist bioactivity was assessed. Table B1-3 summarizes theTLR7/8 agonist bioactivity relationships for Compound Nos. 63-00, 63-33,63-35, 63-36, and 63-38 through 63-47. The TLR7 and TLR8 agonist potencyshown in Table B1-3 is reported as a percent of the effective compoundconcentration at 50% of the maximal response determined for Compound No.63-00. Variation between human blood donors in the levels of cytokinessecreted from purified immune cells used to assess the potency of TLR7and TLR8 agonist compounds results in minor variations in the absolutevalues calculated for EC₅₀ potency for a given compound; to normalizefor this effect, the data in Table B1-3 is expressed as a percentage ofthe EC₅₀ determined for the unmodified, imidazoquinoline-based chemicalstructure (Compound No. 63-00).

As shown in Table B1-3, modification of the chemical structure ofCompound No. 63-00 to produce compounds of formula (J-1) with varyingcycloalkyl moieties resulted in attenuation of the TLR7 agonist potencyup to 11-fold. The (cyclobutyl)methyl (Compound No. 63-44) and(1-methylcyclobutyl)methyl (Compound No. 63-45) variants demonstratedthe least amount of attenuated TLR7 agonist activity (1.2 and 1.9-foldlower potency, respectively), whereas the (cyclopentyl)ethyl (CompoundNo. 63-36) and (2-cyclobutyl)-(2,2-dimethyl)ethyl (Compound No. 63-47)variants demonstrated the greatest amount of attenuated TLR7 agonistactivity (10.9 and 9.0-fold lower potency, respectively). Unexpectedly,the same set of structural modifications to the chemical structure ofCompound No. 63-00 resulted in comparable or greater TLR8 agonistpotency. The (cyclobutyl)methyl (Compound No. 63-44) and(1-methylcyclobutyl)methyl (Compound No. 63-45) variants demonstratedthe greatest increase in TLR8 agonist potency (5.9 and 7.7-fold greaterpotency, respectively). In contrast, the (1-methylcyclopropyl)ethyl(Compound No. 63-42) and (2-cyclobutyl)-(2,2-dimethyl)ethyl (CompoundNo. 63-47) variants demonstrated slightly improved to slightly inferiorTLR8 agonist activity compared to Compound No. 63-00. A TLR7/8 agonistsmall molecule with more closely matched TLR7 and TLR8 agonist potencyis more likely to yield comparable activation of the 2 receptors systemsupon administration of a therapeutic dose of compound in a givenpharmaceutical composition, thus activating a broader range of relevantimmune cell types. Compounds with balanced dual potency would also allowfor the synthesis and characterization of a single active pharmaceuticalingredient, thereby facilitating GMP manufacturing at lower costs andenabling a more straightforward and predictable regulatory pathway.

TABLE B1-3 Structural and Bioactivity Relationships for Select Compoundsof Formula (J-1). TLR7 TLR8 (% of Compound No. (% of Compound No.Compound No. 63-00 EC₅₀ Value) 63-00 EC₅₀ Value) 63-33 290 41 63-35 71553 63-36 1,085 72 63-38 345 37 63-39 220 48 63-40 215 51 63-41 360 4363-42 445 85 63-43 440 77 63-44 120 17 63-45 190 13 63-46 400 39 63-47900 108 63-00 100 100

Example B2. Preparation of Pharmaceutical Compositions

Example B2-1. Preparation of sesame oil-based pharmaceuticalcompositions. Compound Nos. 63-17, 63-18, 63-10, and 63-33 wereformulated for in vivo administration in 95% sesame oil/5% ethanol (v/v)as follows. Super Refined® sesame oil was obtained from Croda Inc.(Edison, N.J.) and ethanol (200 proof, USP grade) was obtained fromPharmaco-AAPER (Brookfield, Conn.). The compounds were placed in a glassvial and 100% ethanol added to make 2.75 mg/mL suspensions. Thesolutions were solubilized by vortexing for 30 seconds and then held inan ultrasonic water bath set at 50° C. for 30 minutes. One mL of thesesolutions was then transferred to a 20 mL glass vial containing 16.0 gof sesame oil, mixed on an end-over-end mixer for 20 minutes at ambienttemperature, and then transferred to a 90° C. water bath for 2 hours toensure complete solubilization. The formulated compounds were cooled to37° C. before sterilization by 0.2 micron filtration. Formulatedcompounds were stored at 2-8° C. in rubber stopper-capped sterile glassvials. The concentrations of the components in the final formulationwere as follows: 0.1 mg/mL (w/v) compound in 95% sesame oil and 5%ethanol (v/v).

Example B2-2. Preparation of squalene oil-in-water nanoemulsion-basedpharmaceutical compositions. Compound Nos. 63-17, 63-18, 63-10, and63-33 were formulated for in vivo administration in squaleneoil-in-water-based nanoemulsions as follows. Squalene (≥98%, liquid),Tween®80 (Polysorbate 80), glycerol, and sodium citrate tribasicdihydrate were obtained from Sigma-Aldrich (St. Louis, Mo.).1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was obtained from AvantiPolar Lipids (Alabaster, Ala.). Cell culture grade water (sterile waterfor injection) was obtained from Corning Life Sciences (Tewksbury,Mass.). To form the oil phase, DOPC (175.6 mg) was added to squalene oil(1.4 mL) in a 4 mL glass vial. The mixture was then incubated in asonicating water bath at 70° C. for 45 minutes, with brief vortexingevery 15 minutes, until the lipid was dissolved. The indicated compound(13.7 mg) was added to the squalene/DOPC solution and vortexedvigorously for one minute. This solution was then incubated in asonicating water bath at 70° C. for 30 minutes, with brief vortexingevery 10 minutes. If needed in order to generate a clear solution, themixture was further incubated in a 90° C. water bath for 2 hours withbrief vortexing every 15 minutes. Separately, to form the water phase,Tween 80 (70 mg) and glycerol (315 mg) were mixed with 100 mM sodiumcitrate pH 6.5 solution (3.5 mL) and water (29.8 mL) in a 50 mLpolypropylene tube.

An oil-in-water emulsion was formed by combining the squaleneoil/DOPC/compound-containing oil phase and the Tween®80/glycerol/sodiumcitrate-containing water phase followed by high shear mixing with aPolytron® mixer (Kinematica, Luzern CH) for 5 minutes at 24,000 rpm. Thecrude emulsion was then submitted to high pressure homogenization usinga Microfluidics M-110P Microfluidizer® (Westwood, Mass.) for 8 passes atapproximately 30,000 psi. Analysis by dynamic light scattering (MalvernNanoS®, Malvern UK) indicated a mean oil drop diameter of 150-175 nm,with a dispersity index of <0.15. The compound-containing nanoemulsionformulation was then sterile filtered using a 0.2 micron sterile syringefilter and stored at 2-8° C. in rubber stopper-capped sterile glassvials.

The concentrations of various components in the final nanoemulsionformulation were as follows: 0.4 mg/mL compound (w/v), 4% squalene oil(v/v), 0.5% DOPC (w/v), 0.2% Tween®80 (w/v), 0.9% glycerol (w/v), and 10mM sodium acetate. Pharmaceutical preparations for the in vivoadministration of the compounds in a squalene oil-in-water nanoemulsionformulation were prepared prior to use by diluting 1:1 in Dulbecco'sphosphate-buffered saline, with mixing by gentle inversion.

Example B3. In Vivo Systemic Immune Activation Assay

Small molecule TLR7 and TLR7/8 agonists derived from the1H-imidazo[4,5-c]quinoline privileged template (see e.g., Imiquimod,Resiquimod) are known to rapidly distribute to the systemic compartmentfollowing intratumoral, subcutaneous, or intramuscular injection. Broadsystemic distribution of these agonist compounds in wild-type miceinduces TLR7-dependent cytokine responses, primarily in spleen and livercells, which can subsequently be detected in the serum within 3-6 hours.The rapid increase in serum cytokine biomarkers (e.g., IL-6 andIL-12p40) can be used to assess the kinetics of systemic distribution ofa locally administered TLR agonist.

The kinetics of distribution of pharmaceutical compositions comprised ofCompound Nos. 63-00, 63-17, or 63-10 formulated in 95% sesame oil/5%ethanol (v/v) were assessed following a single subcutaneous injection inwild-type mice. All in vivo procedures were conducted in accordance withapproved Institutional Animal Care and Use Committee (IACUC) protocols.The animals were housed in a facility that is accredited by theAssociation for Accreditation and Laboratory Animal Care (AALAC,Frederick Md.). Wild-type female BALB/c mice (15-20 gm) were obtainedfrom Envigo (Hayward, Calif.) and acclimated for 2-3 days prior to use.

The pharmaceutical compositions were made with the three compounds, at afinal concentration of 200 μg/mL, in a manner similar to that describedin Example B2-1. At T=0, groups of 3 mice were anesthetized with 1%isoflurane and injected subcutaneously in the right footpad with 5 μg ofeach of the three compounds, or a vehicle control, in 25 uL of 95%sesame oil/5% ethanol (v/v). Then, at T=3, 6, and 24 hours, threeanimals from each group were anesthetized with 1% isoflurane tofacilitate blood collection by cardiac puncture. Samples were processedto serum, and stored at −20° C. for further analysis.

Serum IL-6 and IL-12p40 levels were quantitated by ELISA from eachindividual animal to determine whether compounds derivatized with longeralkyl chains demonstrated a slower rate of systemic distribution. Thenon-alkyl chain modified Compound No. 63-00 induced elevated serumlevels of IL-6 and IL-12p40 at 3 hours (IL-6=772±141 pg/mL,IL-12p40=139,767±31,024 pg/mL) and 6 hours (IL-6=160±19 pg/mL,IL-12p40=142,359±22,350 pg/mL) which returned to baseline by 24 hours(IL-6=32±2 pg/mL, IL-12p40=7,796±1,545 pg/mL), as shown in FIG. 1. Thepentylamino variant, Compound No. 63-17, showed a similar magnitude andkinetics of initial cytokine production in the serum compartment at 3hours (IL-6=853±539 pg/mL, IL-12p40=84,731±32,530 pg/mL) and at 6 hours(IL-6=414±105 pg/mL, IL-12p40=258,645±19,982 pg/mL), as well as a returnto baseline levels by 24 hours (IL-6=33±4 pg/mL, IL-12p40=19,546±2,116pg/mL). The tetradecanamide variant, Compound No. 63-10, which possesseda substantially higher cLogP value, demonstrated no detectable increasein serum cytokines at 3, 6, or 24 hours (3 hours: IL-6=49±32 pg/mL,IL-12p40=1,012±246 pg/mL; at 6 hours: IL-6=33±4 pg/mL,IL-12p40=2,179±597 pg/mL; and at 24 hours: IL-6=32±2 pg/mL,IL-12p40=2,436±237 pg/mL). These data are consistent with theinterpretation that despite the fact that all three compoundsdemonstrate equal TLR7 agonist bioactivity in vitro (see Tables B1-1 andB1-2) the higher hydrophobicity of Compound No. 63-10, as well as itsformulation in a pharmaceutical composition of sesame oil/ethanolpromotes the molecule's retention at the site of injection.

Example B4. Anti-Tumor Efficacy of Alky Chain Modified TLR7/8 Agonistsin CT26 Colon Carcinoma Bearing Wild-Type Mice

The effect of repeated weekly doses of intratumorally deliveredpharmaceutical compositions comprised of Compound No. 63-10, 63-18, or63-33 formulated in 95% sesame oil/5% ethanol (v/v) on tumor growthinhibition was assessed in syngeneic CT26 colon carcinoma-bearing Balb/cmice. All in vivo procedures were conducted in accordance with approvedInstitutional Animal Care and Use Committee (IACUC) protocols. Theanimals were housed in a facility that is accredited by the Associationfor Accreditation and Laboratory Animal Care (AALAC, Frederick, Md.).Wild-type female Balb/c mice (15-20 gm) were obtained from Envigo(Hayward, Calif.) and acclimated for 2-3 days prior to use.

The pharmaceutical compositions were made with the three compounds atfinal concentrations of 5, 50, and 200 μg/mL in a manner similar to thatdescribed in Example B2-1. A TLR9 CpG agonist that has previouslydemonstrated efficacy in this murine tumor model was used as a positivecontrol (Wang et al 2016 PNAS 113:E7240-E7249). On day 0, mice wereanesthetized with 1% isoflurane and 80,000 CT26 tumor cells in 200 uL ofRMPI-1640 culture media plus 2.5% fetal bovine serum were injectedsubcutaneously in the right flank. Tumors were allowed to grow untilthey were ˜35 mm³, at which point animals were assigned to groups tobegin treatment. Mice were injected weekly for 4 weeks intratumorallywith 100 uL of a pharmaceutical composition comprising 20, 5, or 0.5 μgsof Compound No. 63-10, or 20 or 5 μgs of Compound Nos. 63-18, or 63-33formulated in 95% sesame oil/5% ethanol (v/v), or a vehicle control,twice weekly for 3 weeks (experimental days 9, 12, 16, 19, 23, and 26).The TLR9 CpG agonist was injected intratumorally with 100 μL of apharmaceutical composition comprised of 50 μgs of compound formulated inphosphate buffered saline on the same dosing schedule. Tumor sizes weremeasured twice weekly from days 8 through day 30 with calipers, withtumor volumes calculated using the formula: length, multiplied by width,multiplied by width, divided by 2.

Compound Nos. 63-10, 63-18, and 63-33 demonstrated robust CT26 tumorgrowth control over the range of doses tested compared to the vehiclecontrol (FIG. 2). The level of tumor growth control observed forCompound Nos. 63-10, 63-18, and 63-33 was comparable to that of the TLR9CpG. These data demonstrate that the TLR7/8 agonists of the currentinvention possess potent anti-tumor effects in this syngeneic mousetumor growth model system that show greater tumor growth controlcompared to the vehicle control (and are comparable to tumor growthinhibition by TLR9 CpGs). These data are consistent with theinterpretation that tumor growth inhibition for Compound Nos. 63-10,63-18, and 63-33 correlates with their TLR7 agonist bioactivity in vitro(see e.g., Tables B1-1 and B1-2) and is independent of their alkyl chainlength modifications.

Example B5. Anti-Tumor Efficacy of Alky Chain Modified TLR7/8 AgonistsCo-Administered with Tumor Associated Antigens in Two Flank CT26 ColonCarcinoma-Bearing Wild-Type Mice

The effect of repeated weekly doses of intratumorally-deliveredpharmaceutical compositions comprised of Compound No. 63-10 formulatedin a squalene-based oil-in-water nanoemulsion that was co-formulatedwith the CT-26 tumor associated AH-1 class II peptide (immunodominantepitope sequence from the endogenous retroviral gene product gp70; seee.g., Rice J, Buchan S and Stevenson F 2002 J Immunol 169:3908-3913) oninjected and distal tumor growth inhibition was assessed in CT26 coloncarcinoma-bearing Balb/c mice bearing tumors in 2 flanks. All in vivoprocedures were conducted in accordance with approved IACUC protocols.The animals were housed in a facility that is accredited by the AALAC.Wild-type female Balb/c mice (15-20 g) were obtained from Envigo(Hayward, Calif.) and acclimated for 2-3 days prior to use.

Pharmaceutical compositions comprised of squalene-based oil-in-waternanoemulsions were made generally as described in Example B2-2. Inaddition to a control squalene-based oil-in-water nanoemulsion,additional nanoemulsions were made that incorporated either Compound No.63-10 alone at a 500 ng/mL final concentration, or Compound No. 63-10 ata 500 ng/mL final concentration plus the AH-1 class II peptide at a500,000 ng/mL final concentration. For the latter pharmaceuticalcomposition, the AH-1 peptide was initially dissolved at 2×concentration in phosphate buffered saline, then formulated into thesqualene-based oil-in-water nanoemulsion during the final mixing step toyield a final concentration in the nanoemulsion of 500,000 ng/mL. On day0, mice were anesthetized with 1% isoflurane, and 80,000 CT26 tumorcells in 200 uL of RMPI-1640 culture media with 2.5% fetal bovine serumwere injected subcutaneously in both the right and left flanks. Tumorswere allowed to grow until day 8, when the average tumor sizes hadreached approximately 50 mm³, at which time mice were randomized intogroups and injected intratumorally in the right flank tumor with 100 uLof the squalene-based oil-in-water nanoemulsion vehicle control,nanoemulsion containing 50 ng of Compound No. 63-10, or nanoemulsioncontaining 50 ng of Compound No. 63-10 plus 50,000 ng of AH-1 tumorantigen peptide. These three pharmaceutical compositions were furtherinjected into the right flank tumor on experimental days 12, 16, and 20.Right (injected) and left (distal) tumor volumes were then measuredtwice weekly from days 8 through day 30 with calipers, with tumorvolumes calculated using the formula: length, multiplied by width,multiplied by width, divided by 2.

The pharmaceutical composition comprised of a squalene-basedoil-in-water nanoemulsion containing 50 ng of Compound Nos. 63-10demonstrated a trend towards greater tumor growth inhibition in theinjected (right) tumor compared to the nanoemulsion vehicle control(FIG. 3A); however, this tumor growth inhibition was not significantlydifferent from the vehicle control at day 27. In contrast, thepharmaceutical composition comprised of a squalene-based oil-in-waternanoemulsion containing 50 ng of Compound Nos. 63-10 plus 50,000 ng ofAH-1 tumor associated peptide demonstrated significantly greater tumorgrowth inhibition in the injected (right) tumor compared to thenanoemulsion vehicle control at day 27. Additionally, the pharmaceuticalcomposition comprised of a squalene-based oil-in-water nanoemulsioncontaining 50 ng of Compound Nos. 63-10 demonstrated a trend towardsgreater tumor growth inhibition in the distal (left) tumor compared tothe nanoemulsion vehicle control (FIG. 3B); however, this tumor growthinhibition was not significantly different from the vehicle control atday 23. In contrast, the pharmaceutical composition comprised of asqualene-based oil-in-water nanoemulsion containing 50 ng of CompoundNos. 63-10 plus 50,000 ng of AH-1 tumor associated peptide demonstratedsignificantly greater tumor growth inhibition in the distal (left) tumorcompared to the nanoemulsion vehicle control at day 23. These data areconsistent with the interpretation that injected and distal tumor growthinhibition by Compound No. 63-10 is superior when it is co-delivered toantigen presenting cells in the tumor microenvironment with anexogenously added CT26 tumor-associated antigen.

Example B6. Anti-Tumor Efficacy of Alky Chain Modified TLR7/8 Agonistsin Combination with Immune Checkpoint Inhibition in Dual Flank CT26Colon Carcinoma-Bearing Wild-Type Mice

The effect of intratumorally delivered pharmaceutical compositionscomprised of Compound No. 63-10 or 63-33 formulated in 95% sesame oil/5%ethanol (v/v), or Compound No. 63-00 formulated in phosphate-bufferedsaline, given in combination with intraperitoneally delivered anti-mousePD-1 (CD279) antibody (an immune checkpoint inhibitor; Bio X Cell,Lebanon N.H.), on tumor growth inhibition was assessed in CT26 coloncarcinoma-bearing Balb/c mice bearing tumors in 2 flanks. All in vivoprocedures were conducted in accordance with approved IACUC protocols.The animals were housed in a facility that is accredited by the AALAC.Wild-type female Balb/c mice (15-20 g) were obtained from Envigo(Hayward, Calif.) and acclimated for 2-3 days prior to use.

The pharmaceutical compositions were made using Compound No. 63-10 or63-33 at final concentrations of 50,000 ng/mL in a manner similar tothat described for Example B2-1. The pharmaceutical composition ofCompound No. 63-00 was made using phosphate-buffered saline at a finalconcentration of 50,000 ng/mL. On day 0, mice were anesthetized with 1%isoflurane, and 80,000 CT26 tumor cells in 200 uL of RMPI-1640 culturemedia plus 2.5% fetal bovine serum were injected subcutaneously in boththe right and left flanks. Tumors (left and right) were allowed to growuntil day 8, when the right and left flank tumor sizes had reachedapproximately 35 mm³, at which point mice were injectedintraperitoneally with 250 μg of anti-PD-1 antibody formulated inphosphate buffered saline or a phosphate buffered saline vehiclecontrol. The anti-PD-1 treatments were repeated on experimental days 12,15, 19, 22, and 26. On experimental day 14, when the right (injected)and left (distal) flank tumors had reached approximately 100 mm³, micewere randomized into treatment groups. The anti-PD-1 plus treatmentgroups were additionally injected intratumorally in the right flanktumor only with 100 uL of a pharmaceutical composition comprising 5,000ng of Compound No. 63-00 in phosphate buffered saline, 5,000 ng ofCompound No. 63-10 in 95% sesame oil/5% ethanol (v/v), or 5,000 ng ofCompound No. 63-33 in 95% sesame oil/5% ethanol (v/v). Tumor sizes weremeasured twice weekly from days 14 through day 29 with calipers, withtumor volumes calculated using the formula: length, multiplied by width,multiplied by width, divided by 2.

The pharmaceutical compositions comprised of 5,000 ng of Compound No.63-00, 63-10, or 63-33 in combination with anti-PD-1 treatmentdemonstrated greater tumor growth inhibition in the injected tumor(right flank) compared to the anti-PD-1 treatment alone (FIG. 4A). Thistumor growth inhibition was significantly different from the anti-PD-1treatment control at day 29 for Compound Nos. 63-33 and 63-00.Additionally, the pharmaceutical compositions comprised of 5,000 ng ofCompound Nos. 63-00 or 60-33 in combination with anti-PD-1 treatmentdemonstrated a trend towards greater tumor growth inhibition in thedistal tumor (left flank) compared to the anti-PD-1 treatment alone(FIG. 4B), although this tumor growth inhibition did not reachstatistical significance from the anti-PD-1 treatment control at day 29.The pharmaceutical composition comprised of 5,000 ng of Compound No.63-10 in combination with anti-PD-1 treatment demonstrated noimprovement in distal tumor growth inhibition compared to the anti-PD-1treatment alone. These data are consistent with the interpretation thatCompound No. 63-33, in combination with the immune checkpoint inhibitoranti-PD-1, is superior at controlling both injected and distal CT26tumor growth compared to treatment with vehicle control plus anti-PD-1.

All publications, including patents, patent applications, and scientificarticles, mentioned in this specification are herein incorporated byreference in their entirety for all purposes to the same extent as ifeach individual publication, including patent, patent application, orscientific article, were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is apparent to those skilled in the art that certainminor changes and modifications will be practiced in light of the aboveteaching. Therefore, the description and examples should not beconstrued as limiting the scope of the invention.

What is claimed is:
 1. A method of treating cancer in a mammaliansubject in need thereof, comprising administering to the mammaliansubject a pharmaceutical composition in an amount sufficient to treatcancer in the mammalian subject, the composition comprising (i) acompound of formula (J), or a salt thereof, and (ii) a pharmaceuticallyacceptable excipient, wherein the compound of formula (J) has thefollowing structure:

wherein: R⁰ is —(CH₂)_(z)(C(CH₃)₂)R^(A) or —(CH₂)_(m)R^(A); m is 0, 1,2, or 3; z is 1 or 2; R^(A) is C₃-C₈ cycloalkyl optionally substitutedby 1 to 4 groups independently selected from the group consisting ofC₁-C₄ alkyl, C₁-C₄ alkylene, and halogen; X is —NH—; R¹ is C₃-C₆ alkyl,—(CH₂)_(p)OR^(1a), —(CH₂)_(p)NHR^(1b) or —(CH₂)_(p)R^(1c); where R^(1a)and R^(1b) are independently C₁-C₃ alkyl; R^(1c) is C₃-C₄ cycloalkyl;and p is 1 or 2; R² is NHR^(2a); where R^(2a) is H, OH, NH₂, or methyl;each R³ is independently halogen, C₁-C₈ alkyl, —(C₁-C₇ alkylene)—NH₂, or—CH₂-phenylene-CH₂NH₂; q is 0, 1, 2, 3, or 4; and R^(4a) and R^(4b) areindependently H or C₁-C₈ alkyl.
 2. The method of claim 1, wherein thepharmaceutical composition is administered by intratumoral injection. 3.The method of claim 1, further comprising administering an effectiveamount of a second therapeutic agent to the subject.
 4. The method ofclaim 3, wherein the second therapeutic agent is a chemotherapeuticagent.
 5. The method of claim 3, wherein the second therapeutic agent isan antagonist of an inhibitory immune checkpoint molecule.
 6. The methodof claim 3, wherein the second therapeutic agent is an epigeneticmodulator.
 7. The method of claim 3, wherein the second therapeuticagent is an inducer of immunogenic cell death.
 8. The method of claim 5,wherein the inhibitory immune checkpoint molecule is selected from thegroup consisting of PD-1, PD-L1, PD-L2, CTLA-4 (CD152), LAG-3, TIM-3,TIGIT, IL-10, and TGF-beta.
 9. The method of claim 1, wherein R⁰ is—(CH₂)_(m)R^(A).
 10. The method of claim 9, wherein m is
 2. 11. Themethod of claim 1, wherein R⁰ is —(CH₂)_(z)(C(CH₃)₂)R^(A).
 12. Themethod of claim 11, wherein z is
 1. 13. The method of claim 9, whereinR^(A) is cyclopropyl, cyclobutyl, or cyclopentyl.
 14. The method ofclaim 9, wherein R^(A) is C₃-C₆ cycloalkyl optionally substituted by 1to 3 groups independently selected from the group consisting of methyl,methylene, and halogen.
 15. The method of claim 1, wherein m is 1 or 2.16. The method of claim 15, wherein R^(A) is C₃-C₈ cycloalkyl.
 17. Themethod of claim 15, wherein R^(A) is cyclopropyl optionally substitutedby 1 to 3 groups independently selected from the group consisting ofmethyl and methylene.
 18. The method of claim 1, wherein m is 0 andR^(A) is cyclohexyl optionally substituted by 1 to 3 groupsindependently selected from the group consisting of methyl andmethylene.
 19. The method of claim 1, wherein R⁰ is selected from thegroup consisting of:


20. The method of claim 1, wherein the compound of formula (J) isCompound No. 63-33, 63-35, 63-36, or 63-38 to 63-49: Compound No.Formula 63-33

63-35

63-36

63-38

63-39

63-40

63-41

63-42

63-43

63-44

63-45

63-46

63-47

63-48

63-49

or a salt thereof.
 21. The method of claim 20, wherein thepharmaceutical composition further comprises an antigen.